Truncated aggrecanase molecules

ABSTRACT

Truncated aggrecanase proteins and nucleotides sequences encoding them as well as processes for producing them are disclosed. Additionally, aggrecanases with amino acid mutations that lead to increased stability and expression levels in comparison with wild-type or native aggrecanases are also disclosed. Aggrecanases of the invention are especially useful for development of compositions for treatment of diseases such as osteoarthritis. Methods for developing inhibitors of the aggrecanase enzymes and antibodies to the enzymes for treatment of conditions characterized by the degradation of aggrecan are also disclosed.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.10/358,283, filed Feb. 5, 2003, now U.S. Pat. No. 7,150,983 which claimspriority to and the benefit of U.S. Provisional Patent Application No.60/354,592, filed Feb. 5, 2002, the entire disclosures of which arehereby incorporated by reference herein for all purposes.

FIELD OF THE INVENTION

The present invention relates to the discovery of truncated aggrecanasemolecules, processes for producing them, and methods employing thesemolecules to develop inhibitors to aggrecanase enzymes. The inventionfurther relates to the development of inhibitors of, as well asantibodies to, aggrecanase enzymes. These inhibitors and antibodies maybe useful for the treatment of various aggrecanase-associated conditionsincluding osteoarthritis.

BACKGROUND OF THE INVENTION

Aggrecan is a major extracellular component of articular cartilage. Itis a proteoglycan responsible for providing cartilage with its-mechanical properties of compressibility and elasticity. The loss ofaggrecan has been implicated in the degradation of articular cartilagein arthritic diseases such as osteoarthritis.

Osteoarthritis is a debilitating disease which affects at least 30million Americans (MacLean et al., J Rheumatol 25:2213–2218 (1998)).Osteoarthritis can severely reduce quality of life due to degradation ofarticular cartilage and the resulting chronic pain. An early andimportant characteristic of the osteoarthritic process is loss ofaggrecan from the extracellular matrix (Brandt and Mankin, “Pathogenesisof Osteoarthritis,” Textbook of Rheumatology, WB Saunders Company,Philadelphia, Pa., pgs. 1355–1373 (1993)). The large, sugar-containingportion of aggrecan is thereby lost from the extra-cellular matrix,resulting in deficiencies in the biomechanical characteristics of thecartilage.

A proteolytic activity termed “aggrecanase” is believed to beresponsible for the cleavage of aggrecan, thereby having a role incartilage degradation associated with osteoarthritis and inflammatoryjoint disease. Research has been conducted to identify the enzymesresponsible for the degradation of aggrecan in human osteoarthriticcartilage. Aggrecan contains two N-terminal globular domains, G1 and G2,separated by a proteolytically sensitive interglobular domain, followedby a glycosaminoglycan attachment region and a C-terminal globulardomain, G3. At least two enzymatic cleavage sites have been identifiedwithin the interglobular domain of aggrecan. One enzymatic cleavage sitewithin the interglobular domain of aggrecan (Asn³⁴¹-Phe³⁴²) has beenobserved to be cleaved by several known metalloproteases. Flannery etal., J Biol Chem 267:1008–1014 (1992); Fosang et al., Biochemical J.304:347–351 (1994). Cleavage at a second aggrecan cleavage site withinaggrecan (Glu³⁷³-Ala³⁷⁴) due to IL-1 induced cartilage aggrecan cleavageresults in the generation of an aggrecan fragment found in humansynovial fluid (Sandy et al., J Clin Invest 89:1512–1516 (1992);Lohmander et al., Arthritis Rheum 36: 1214–1222 (1993); Sandy et al., JBiol Chem 266: 8683–8685 (1991)). Aggrecan cleavage at (Glu³⁷³-Ala³⁷⁴)has been attributed to aggrecanase activity (Sandy et al., J Clin Invest89:1512–1516 (1992). This Glu³⁷³-Ala³⁷⁴ cleavage site will be referredto as the aggrecanase cleavage site.

Recently, identification of two enzymes, aggrecanase-1 (ADAMTS4) andaggrecanase-2 (ADAMTS-11) within the “A Disintegrin andMetalloproteinase with Thrombospondin motifs” (ADAMTS) family, have beenidentified which are synthesized by IL-1 stimulated cartilage and cleaveaggrecan at the Glu³⁷³-Ala³⁷⁴ site (Tortorella et al., Science284:1664–1666 (1999); Abbaszade et al., J Biol Chem 274: 23443–23450(1999)). Aggrecanase-1 is reported to include at least six domains:signal; propeptide; catalytic; disintegrin; TSP type-1 motif andC-terminal. Aggrecanase-2 is also a multidomain protein. It is reportedto have a signal sequence; a prodomain; a metalloproteinase domain; adisintegrin domain and a spacer domain between a TSP motif and a TSP submotif in the C-terminal of the protein. It was generally believed thatthe TSP domains and the spacer domain are critical for substraterecognition. Specifically, Tortorella et al. reported that “[T]hisregion may serve to bind aggrecanase-1 to the glycosaminoglycans of theaggrecan substrate.” See Tortorella et al., Science 284:1664–1666(1999).

It is contemplated that there are other, related enzymes in the ADAMTSfamily which are capable of cleaving aggrecan at the Glu³⁷³-Ala³⁷⁴ bondand could contribute to aggrecan cleavage in osteoarthritis. It ispossible that these enzymes could be synthesized by osteoarthritic humanarticular cartilage. However, it has been difficult to developinhibitors and treatment therapies to treat diseases that involveaggrecan cleavage because aggrecanases have been difficult to isolateand purify in large amounts due to poor stability of these molecules andgenerally low expression levels. Therefore, there is a need to identifynovel forms of aggrecanases and further develop ways to isolate andpurify aggrecanase proteins in large amounts in order to investigatetheir role in disease states and also to develop therapies andcompositions to treat diseases involving aggrecan cleavage.

SUMMARY OF THE INVENTION

The present invention is directed to truncated aggrecanase proteins andvariants and fragments thereof; nucleotide sequences which encodetruncated aggrecanase enzymes of the invention and fragments andvariants thereof; and processes for the production of truncatedaggrecanases. Truncated aggrecanases of the invention are biologicallyactive and have greater stability and higher expression levels thantheir full-length counterparts. More specifically, the inventionfeatures truncated aggrecanase-1 and aggrecanase-2 enzymes that are morestable and show higher levels of expression than full-lengthaggrecanase-1 and aggrecanase-2 enzymes, respectively; nucleic acidsequences encoding truncated aggrecanases-1 and 2 of the invention andfragments and variants thereof; and methods of producing truncatedaggrecanases 1 and 2, or fragments and variants thereof.

In one embodiment, truncated aggrecanases of the invention compriseaggrecanases that have at least one TSP domain deleted. In anotherembodiment, truncated aggrecanases of the invention compriseaggrecanases that have at least two TSP domains deleted. Although, TSPdomains in aggrecanases have been thought to be important for substraterecognition, and therefore, for the ability of aggrecanase to recognizeand subsequently cleave aggrecan, truncated aggrecanase proteins of theinvention are biologically active despite deletion of one or both TSPdomains in the proteins.

Truncated aggrecanases of the invention have greater stability and areexpressed at higher levels compared with the full-length aggrecanaseproteins, thereby facilitating isolation, purification, and use ofaggrecanases of the invention in the development of inhibitors andtherapies for treatment of diseases. Accordingly, in one embodiment, theinvention comprises methods for producing large amounts of purifiedtruncated aggrecanases that may be used for development of inhibitorsand treatment therapies.

The invention further includes compositions comprising truncatedaggrecanases of the invention and use of such compositions for thedevelopment of inhibitors of aggrecanases for treatment of diseasesincluding osteoarthritis. In addition, the invention includes methodsfor identifying and developing inhibitors of aggrecanase which block theenzyme's activity. The invention also includes antibodies to theseenzymes, in one embodiment, for example, antibodies that blockaggrecanase activity. These inhibitors and antibodies may be used invarious assays and therapies for treatment of conditions characterizedby the degradation of articular cartilage. In one embodiment, inhibitorsare peptide molecules that bind aggrecanases.

This invention provides amino acid sequences of biologically activetruncated aggrecanase molecules that have greater stability comparedwith the full-length aggrecanase protein.

In one aspect, the invention features biologically active truncatedaggrecanase-2 molecules that have at least one TSP domain deleted, suchas a protein with an amino acid sequence from amino acid #1 (Met)through amino acid #753 (Glu) of SEQ ID NO: 4; from amino acid #1 (Met)through amino acid #752 (Pro) of SEQ ID NO: 6; and from amino acid #1(Met) through amino acid #628 (Phe) of SEQ ID NO: 8, and variants andfragments thereof, including substitution mutants, that exhibitaggrecanase activity.

The invention also features truncated aggrecanase-1 molecules that haveat least one TSP domain deleted. An example includes an aggrecanase-1protein with an amino acid sequence from amino acid #1 (Met) throughamino acid #520 (Ala) of SEQ ID NO: 13, fragments, and variants thereofincluding substitution mutants that exhibit aggrecanase activity.

In another embodiment, the invention features biologically activetruncated aggrecanase-2 proteins with at least two TSP domains deletedcomprising, for example, a protein with an amino acid sequence fromamino acid #1 through amino acid #527 (His) of SEQ ID NO: 10 (FIG. 10),variants, and fragments thereof including substitution mutants thatexhibit aggrecanase activity.

Truncated aggrecanases with one or both TSP domains deleted arebiologically active and are more stable than the full-length aggrecanaseenzymes.

The invention also features nucleic acid molecules that encode truncatedaggrecanases of the invention. For example, nucleic acid moleculesencoding truncated aggrecanase-2 molecules of the invention include:nucleotide #1 through nucleotide #2259 of SEQ ID NO: 3 (FIG. 3), whichencodes a polypeptide set forth in SEQ ID NO: 4; nucleotide #1 throughnucleotide #2256 of SEQ ID NO: 5 (FIG. 5), which encodes a polypeptideset forth in SEQ ID NO: 6; nucleotide #1 through nucleotide #1884 of SEQID NO: 7 (FIG. 7), which encodes the polypeptide set forth in SEQ ID NO:8; and nucleotide #1 through nucleotide #1701 of SEQ ID NO: 9 (FIG. 9),which encodes the polypeptide set forth in SEQ ID NO: 10. Nucleic acidmolecules of the invention further include fragments and variants of SEQID NOs: 3, 5, 7, and 9 which encode truncated aggrecanase-2 molecules ofthe invention, nucleotide sequences that hybridize under moderate tostringent conditions with nucleotide sequences of SEQ ID NOs: 3, 5, 7,or 9 and fragments or variants thereof, naturally occurring allelicsequences, and equivalent degenerative codon sequences of aggrecanase-2nucleic acid sequences disclosed herein.

Nucleic acid molecules encoding truncated aggrecanase-1 molecules of theinvention include, for example, nucleotides which encode thepolypeptides of SEQ ID NOs: 12 and 13; set forth in FIGS. 23 and 24respectively, fragments and variants of nucleic acids that encodetruncated aggrecanase-1 molecules of the invention, nucleotide sequencesthat hybridize under moderate to stringent conditions to nucleic acidsequences that encode truncated aggrecanase-1 molecules of theinvention, for example, nucleic acid sequences of FIGS. 23 and 24, orfragments and variants thereof, naturally occurring allelic sequencesand equivalent degenerative codon sequences of aggrecanase-1 encodingnucleic acid sequences. A nucleic acid sequence for full-lengthaggrecanase-1 molecule is found in Genbank under Accession No.:NM_(—)005099 (FIG. 22). Therefore, one skilled in the art can use thissequence to generate a full-length aggrecanase-1 molecule set forth inSEQ ID NO: 11, or use part of the nucleotide sequence to generate atruncated protein, for example, the aggrecanase protein set forth inFIG. 12 or 13. For example nucleotide #407 through nucleotide #2132 ofthe published NM_(—)005099 sequence (FIG. 23) (SEQ ID NO: 32) may beused for generating the truncated aggrecanase-1 protein of SEQ ID NO:12. Similarly, nucleotide #1 through nucleotide #1967 of the publishedNM_(—)005099 sequence (FIG. 24) (SEQ ID NO: 33) may be used for thegeneration of truncated aggrecanase molecule of FIG. 13.

In another aspect, the invention includes aggrecanase molecules thatcomprise mutations that increase stability and expression levels oftruncated aggrecanase molecules compared with their full-lengthcounterparts. Aggrecanases with mutations in their active sites areparticularly useful for the synthesis of inhibitors of aggrecanases.Accordingly, in one embodiment, the invention features an aggrecanase-2molecule with a mutation at amino acid 411 (E411-Q411 mutation) in theactive site within the catalytic domain. The amino acid sequence of anaggrecanase-2 molecule with the E411-Q411 mutation is shown in FIG. 21(SEQ ID NO: 30). Mutations that lead to increased stability of theaggrecanase proteins in comparison with their wild-type counterparts canbe made in both truncated as well as full-length aggrecanase proteins.It is contemplated that mutations that alter stability of aggrecanasemolecules can be made within the catalytic domain of an aggrecanaseprotein or outside the catalytic domain. Aggrecanase proteins carryingsuch mutations may be found in nature or may be generated artificially.One skilled in the art can test the effect of a mutation on thestability of an aggrecanase molecule by one of many assays provided.Mutations of the invention include, for example, amino acidsubstitutions or modifications. Amino acid mutations in aggrecanases ofthe invention can be generated by mutagenesis, chemical alteration, orby alteration of DNA sequence used to produce the polypeptide.

Aggrecanase-1 molecules can also be generated to include mutations inthe catalytic domain in order to increase their stability. For example,FIG. 17 features flag-tagged truncated aggrecanase-1 proteins thatinclude an E-to-Q amino acid mutation in the catalytic domain oftruncated aggrecanase-1 molecules, the wild-type sequences of which areset forth in SEQ ID NO: 12 and 13, thereby leading to increasedstability of aggrecanase-1 proteins compared to full-length wild-typeaggrecanase-1 protein, the sequence of which is set forth in SEQ ID NO:11. These aggrecanases are particularly useful for the development aswell as identification of novel inhibitors of aggrecanases.

The invention further features truncated and/or mutant aggrecanasefamily members and aggrecanase-like proteins with deletions and/orsubstitution mutations, where a deletion or an amino acid substitutionmutation occurs in a region of the protein comparable to that ofaggrecanases of the invention.

The invention further includes variants and equivalent degenerativecodon sequences of nucleic acid sequences described herein that encodebiologically active truncated aggrecanase polypeptides. Additionally,the invention includes nucleic acid molecules that hybridize undermoderate to stringent conditions to the nucleic acids of the invention,allelic variants and substitution and deletion mutants of nucleic acidsmolecules described herein. In one embodiment, truncated aggrecanasesand/or aggrecanases carrying at least one amino acid substitutionencoded by nucleic acid molecules of the invention are more stable thanthe corresponding full-length aggrecanase protein and can be expressedat higher levels than the full-length aggrecanase protein. In oneembodiment, mutations are introduced in nucleic acid molecules encodingaggrecanases of the invention that lead to mutations; for example, aminoacid substitutions, in the protein encoded by the nucleic acid carryingthe mutation. One example of such a mutation is a nucleic acid sequenceencoding an aggrecanase-2 protein with an E-to-Q mutation in the activesite of molecule. In another embodiment, the invention includesaggrecanase-1 nucleic acid molecules that encode aggrecanase-1 proteinscomprising an E-to-Q mutation in the catalytic domain, thereby leadingto generation of molecules with greater stability, longer half-lives andincreased levels of expression as compared to the full-lengthaggrecanases 1 and 2.

It is expected that other species have DNA sequences that are similar oridentical to human aggrecanase enzymes described herein. Accordingly,the invention further includes methods for obtaining other nucleic acidmolecules encoding truncated aggrecanases or aggrecanase-like moleculesor aggrecanases with amino acid substitutions that alter theirbiological activity, from humans as well as non-human species. In oneembodiment, a method for isolating a nucleic acid sequence encoding anaggrecanase of the invention involves utilizing a nucleic acid sequencedisclosed herein or variants or fragments thereof; for example, SEQ IDNO: 1 or a fragment or a variant thereof; SEQ ID NO: 3 or a fragment ora variant thereof; SEQ ID NO: 5 or a fragment or a variant thereof; SEQID NO: 7 or a fragment or a variant thereof; SEQ ID NO: 9 or a fragmentor a variant thereof; SEQ ID NO: 31 or a fragment or a variant thereof;SEQ ID NO: 32 or a fragment or variant thereof; or SEQ ID NO: 33 or afragment or a variant thereof, to design probes for screening librariesfor the corresponding gene from other species or coding regions of genesthat encode proteins/peptides with aggrecanase activity. Therefore, theinvention includes DNA sequences from other species, which arehomologous to human aggrecanase sequences, or fragments or variantsthereof, and can be obtained using at least one of the DNA sequencesprovided herein. In addition, the present invention includes DNAsequences that encode fragments or variants of aggrecanases of theinvention. The present invention may also include functional fragmentsof the aggrecanase protein, and DNA sequences encoding such functionalfragments, as well as functional fragments of other related proteins.The ability of such a fragment to function is determinable by an assayof the protein in one of many biological assays described for the assayof the aggrecanase protein.

In another aspect, the invention provides methods for producing isolatedtruncated aggrecanases of the invention. In one embodiment, a humanaggrecanase protein of the invention or a variant or fragment thereofmay be produced, for example, by culturing a cell transformed with a DNAsequence: from nucleotide #1 through nucleotide #2259, set forth in SEQID NO: 3; or from nucleotide #1 through nucleotide #2256, set forth inFIG. 5; or from nucleotide #1 through nucleotide #1884, set forth inFIG. 7; or from nucleotide #1 through nucleotide #1701, set forth inFIG. 9, and recovering and purifying from the culture medium anaggrecanase-2 protein characterized by the amino acid sequence set forthin: FIG. 4 from amino acid #1 (Met) through amino acid #753 (Glu); FIG.6 from amino acid #1 (Met) through amino acid #752 (Pro); FIG. 8 fromamino acid #1 (Met) through amino acid #628 (Phe); or FIG. 10 from aminoacid #1 (Met) through amino acid #567 (His). Similarly, nucleic acidsequences expressing truncated aggrecanase-1 molecules; for example,nucleic acid sequences set forth in FIGS. 23 and 24 can be used for theproduction of truncated aggrecanase-1 proteins set forth in FIGS. 12 and13, where the aggrecanase-1 proteins are substantially free from otherproteinaceous materials with which they are co-produced.

In another embodiment, truncated aggrecanase proteins of the inventionmay be produced by culturing a cell transformed with a full-length DNAsequence for aggrecanase, and recovering truncated aggrecanase proteinsfrom the culture medium. Accordingly, in one embodiment, a truncatedaggrecanase-2 protein including amino acid #1 (Met) to amino acid #753(Glu), set forth in SEQ ID No: 4, is recovered from the culture mediumof a cell transformed with a full-length nucleic acid molecule foraggrecanase-2; for example, nucleic acid molecule set forth in SEQ IDNO: 1. In another embodiment, a truncated aggrecanase-2 moleculeincluding amino acid #1 (Met) to amino acid #752 (Pro), is recoveredfrom culture medium of cells transformed with a nucleic acid moleculefor full-length aggrecanase-2. Truncated aggrecanase protein of SEQ IDNO: 4 results from cleavage of the full-length aggrecanase protein, setforth in SEQ ID. NO: 2, at E⁷⁵³-G⁷⁵⁴, yielding a 55 kDa protein. Thenucleotide and amino acid sequences of the full-length aggrecanase-2molecule are set forth in SEQ ID NO: 1 and SEQ ID NO: 2 respectively(Accession Nos: NM_(—)007038 and NP_(—)008969) (FIGS. 1A–1C and 2).

Truncated aggrecanases of the invention that are purified from a culturemedium are substantially free from other proteinaceous materials. Arecovered purified aggrecanase protein having at least one TSP domaindeleted generally exhibits proteolytic aggrecanase activity by cleavingaggrecan., as disclosed. Therefore, truncated proteins of the inventionmay be further characterized by the ability to demonstrate aggrecanproteolytic activity in an assay which determines the presence of anaggrecan-degrading molecule. These assays or the development thereof iswithin the knowledge of one skilled in the art. Such assays may involvecontacting an aggrecan substrate with a truncated aggrecanase moleculeand monitoring the production of aggrecan fragments (see, for example,Hughes et al., Biochem J 305: 799–804 (1995); Mercuri et al., J. BioChem. 274:32387–32395 (1999)). For production in mammalian cells, a DNAsequence used for expression of a truncated aggrecanase of the inventionfurther comprises a DNA sequence encoding a suitable propeptide 5′ toand linked in frame to the nucleotide sequence encoding the aggrecanaseenzyme.

The invention also provides antibodies that bind to isolated aggrecanaseproteins of the invention. In one embodiment, such an antibody reduces,inhibits or antagonizes aggrecanase activity. The invention furtherprovides methods for developing and identifying inhibitors ofaggrecanase activity comprising the use of a truncated aggrecanaseprotein with amino acid sequence chosen from, for example, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO:13 or a fragment or a variant thereof. In one embodiment, inhibitors ofaggrecanase activity prevent cleavage of aggrecan.

Additionally, the invention provides pharmaceutical compositions forinhibiting the proteolytic activity of aggrecanases, wherein thecompositions comprise at least one antibody according to the inventionand at least one pharmaceutical carrier. The invention also providesmethods for inhibiting aggrecanase activity in a mammal comprisingadministering to the mammal an effective amount of a pharmaceuticalcomposition according to the invention to inhibit aggrecanase activity.

In another embodiment, the invention includes methods for identifying ordeveloping inhibitors of aggrecanases and the inhibitors producedthereby. In one embodiment, inhibitors of the invention prevent bindingof an aggrecanase to an aggrecan molecule. In another embodiment,inhibitors of the invention prevent cleavage of aggrecan by aggrecanase.

The method may entail the identification of inhibitors based on an assaycomprising combining at least one aggrecanase protein of this inventionwith at least one test sample; and determining if the test sampleinhibits activity of the aggrecanase protein. The test sample maycomprise known or unknown samples, and these samples may be peptides,proteins, chemical compounds (often referred to as small molecules), orantibodies. They may be selected for testing individually, or inbatches, such as from a library. The art provides aggrecanase activityassays that could be easily utilized in such a method. Assays forinhibitors may involve contacting a mixture of aggrecan and theinhibitor with an aggrecanase molecule followed by measurement of theaggrecanase inhibition; for instance, by detection and measurement ofaggrecan fragments produced by cleavage at an aggrecanase-susceptiblesite.

The method may also entail the determination of binding sites based onat least one of the amino acid sequences of aggrecanase and thethree-dimensional structure of aggrecanase, and optionally aggrecan.Based on this information, one could develop or identify a candidatemolecule that may inhibit aggrecanase activity based on a structuralanalysis, such as predicted structural interaction with the bindingsite. In one embodiment, such a molecule may comprise a peptide,protein, chemical compound, or antibody. Candidate molecules may belater assayed for actual inhibitory activity of the aggrecanase enzyme,as discussed.

Another aspect of the invention therefore provides pharmaceuticalcompositions containing a therapeutically effective amount ofaggrecanase inhibitors in a pharmaceutically acceptable vehicle.

Aggrecanase-mediated degradation of aggrecan in cartilage has beenimplicated in osteoarthritis and other inflammatory diseases. Therefore,these compositions of the invention may be used in the treatment ofdiseases characterized by the degradation of aggrecan and/or an upregulation of aggrecanase. The compositions may be used in the treatmentof these conditions or in the prevention thereof.

The invention further includes methods for treating patients sufferingfrom conditions characterized by a degradation of aggrecan or preventingsuch conditions. These methods, according to the invention, entailadministering to a patient needing such treatment an effective amount ofa composition comprising an aggrecanase inhibitor which inhibits theproteolytic activity of aggrecanase enzymes.

The DNA sequences of the present invention are useful, for example, asprobes for the detection of mRNA encoding aggrecanase in a given cellpopulation. Thus, the present invention includes methods of detecting ordiagnosing genetic disorders involving aggrecanases, or disordersinvolving cellular, organ, or tissue disorders in which aggrecanase isirregularly transcribed or expressed. The DNA sequences may also beuseful for preparing vectors for gene therapy applications as describedbelow.

A further aspect of the invention includes vectors comprising a DNAsequence as described above in operative association with an expressioncontrol sequence therefor. These vectors may be employed in a novelprocess for producing an aggrecanase protein of the invention in which acell line transformed with a DNA sequence encoding an aggrecanaseprotein in operative association with an expression control sequencetherefor, is cultured in a suitable culture medium and an aggrecanaseprotein is recovered and purified therefrom. This process may employ anumber of known cells, both prokaryotic and eukaryotic in origin, ashost cells for expression of the polypeptide. The vectors may be used ingene therapy applications. In such use, the vectors may be transfectedinto the cells of a patient ex vivo, and the cells may be reintroducedinto a patient. Alternatively, the vectors may be introduced into apatient in vivo through targeted transfection.

Additional aspects of the disclosure will be set forth in part in thedescription, and in part be obvious from the description, or may belearned from practicing the invention. The invention is set forth andparticularly pointed out in the claims, and the disclosure should not beconstrued as limiting the scope of the claims. The following detaileddescription includes exemplary representations of various embodiments ofthe invention, which are not restrictive of the invention as claimed.The accompanying figures constitute a part of this specification and,together with the description, serve to illustrate embodiments and notlimit the invention.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1C show the nucleotide sequence of a full-lengthaggrecanase-2/ADAMTS-5 molecule (SEQ ID NO: 1).

FIG. 2 shows the amino acid sequence of a full lengthaggrecanase-2/ADAMTS-5 molecule (SEQ ID NO: 2) encoded by nucleotide #1through nucleotide #2915 of SEQ ID NO: 1.

FIG. 3 shows the nucleotide sequence of a truncated aggrecanase-2protein of the invention from nucleotide #1 through nucleotide #2259(SEQ ID NO: 3).

FIG. 4 shows the amino acid sequence of a truncated aggrecanase-2protein .of the invention from amino acid #1 through amino acid #753(SEQ ID NO: 4), encoded by nucleotide sequence set forth in SEQ ID NO:3.

FIG. 5 shows the nucleotide sequence of a truncatedaggrecanase-2/ADAMTS-5 of the invention from nucleotide #1 throughnucleotide #2256 (SEQ ID NO: 5).

FIG. 6 shows the amino acid sequence of a truncated aggrecanase-2protein of the invention from amino acid #1 through amino acid #752 (SEQID NO: 6), encoded by nucleotide sequence set forth in SEQ ID NO: 5.

FIG. 7 shows the nucleotide sequence of a truncated aggrecanase-2molecule of the invention from nucleotide #1 through nucleotide #1884(SEQ ID NO: 7).

FIG. 8 shows the amino acid sequence of a truncated aggrecanase-2protein of the invention from amino acid #1 through amino acid #628 (SEQID NO: 8), encoded by the nucleotide sequence set forth in SEQ ID NO: 7.

FIG. 9 shows the nucleotide sequence of a truncated aggrecanase-2molecule of the invention from nucleotide #1 through nucleotide #1701(SEQ ID NO: 9).

FIG. 10 shows the amino acid sequence of a truncated aggrecanase-2protein of the invention from amino acid #1 through amino acid #567 (SEQID NO: 10), encoded by the nucleotide sequence set forth in SEQ ID NO:9.

FIG. 11 shows the amino acid sequence of a full-length aggrecanase-1molecule (SEQ ID NO: 11).

FIG. 12 shows the amino acid sequence of a truncated aggrecanase-1molecule including amino acid #1 through amino acid #575 (Pro) (SEQ IDNO: 12).

FIG. 13 shows the amino acid sequence of a truncated aggrecanase-1molecule including amino acid #1 through amino acid #520 (Ala) (SEQ IDNO: 13).

FIG. 14 shows the nucleotide sequence of a recombinant truncatedaggrecanase-2 protein comprising a peptide linker and a streptavidin tag(SEQ ID NO: 14).

FIG. 15 shows the amino acid sequence of a recombinant truncatedaggrecanase-2 protein (SEQ ID NO: 15), encoded by nucleotide sequenceset forth in SEQ ID NO: 14.

FIG. 16 shows aggrecanase activity of conditioned medium from CHO cellsexpressing wild-type (ADAMTS-5 WT) or active-site mutant (ADAMTS-5 ASM)as detected by the aggrecanase ELISA assay.

FIG. 17 shows a schematic representation of flag-tagged truncatedaggrecanase-1 molecules with an E-to-Q mutation in the catalytic domain.

FIG. 18 shows a schematic representation of streptavidin taggedtruncated aggrecanase-2 molecules.

FIG. 19 shows a western blot with expression of truncated aggrecanase-2proteins in comparison with a full-length aggrecanase-2 protein.

FIG. 20 shows percent aggrecan cleavage by truncated aggrecanase-2molecules of the invention in a 3B3 ELISA assay.

FIG. 21 shows the amino acid sequence for a full-length ADAMTS-5 proteinwith an E-to-Q mutation at amino acid position 411 (SEQ IS NO: 30).

FIGS. 22A and 22B show a nucleic acid sequence encoding a full-lengthaggrecanase-1 protein (SEQ ID NO: 31).

FIG. 23 shows a nucleic acid sequence encoding a truncated aggrecanase-1molecule; for example, the protein set forth in FIG. 12 (SEQ ID NO: 32).

FIG. 24 shows a nucleic acid sequence encoding a truncated aggrecanase-1molecule; for example, the protein set forth in FIG. 13 (SEQ ID NO: 33).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “aggrecanase” refers to a family of polypeptides that arecapable of cleaving the aggrecan protein. Generally, these are proteinsthat cleave aggrecan at the Glu³⁷³-Ala³⁷⁴ aggrecanase cleavage site.Aggrecanases of the present invention encompass but are not limited tothe sequences of SEQ ID NO: 11 (aggrecanase-1) and SEQ ID NO: 2(aggrecanase-2). The term “aggrecanase” includes naturally occurringvariants SEQ ID NOs: 11 and 2, as well as fragments of the sequencesencoded by SEQ ID NOs: 11 and 2 that are active in at least one of theassays provided. For example, included in this definition are amino acidsequences substantially similar or substantially identical to the aminoacid of SEQ ID NOs: 11 or 2 or fragments thereof; or an amino acidsequence at least about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, or about 99% identicalto the amino acid sequence of SEQ ID NO: 11 or 2, or a fragment thereof.

The term aggrecanase further includes the proteins encoded by thenucleic acid sequence of SEQ ID NO: 31 and 1 (aggrecanase-1 and 2respectively) disclosed, fragments and variants thereof. In oneembodiment, the nucleic acids of the present invention will possess asequence which is either derived from, or is a variant of a naturalaggrecanase encoding gene, or a fragment thereof.

The term “antibody” refers to an immunoglobulin, or a fragment thereof,and encompasses any polypeptide comprising an antigen-binding site. Theterm includes but is not limited to polyclonal, monoclonal,monospecific, polyspecific, non-specific, humanized, human,single-chain, chimeric, synthetic, recombinant, hybrid, mutated,grafted, and in vitro generated antibodies. It also includes, unlessotherwise stated, antibody fragments such as Fab, F(ab′)₂, Fv, scFv, Fd,dAb, and other antibody fragments which retain the antigen bindingfunction.

The term “biological activity” refers to at least one cellular processinterrupted or initiated by an aggrecanase enzyme binding to aggrecan.Generally, biological activity refers to proteolytic cleavage ofaggrecan by aggrecanase. Aggrecanase activities include, but are notlimited to, binding of aggrecanase to aggrecan and cleavage of aggrecanby aggrecanase. Activity can also include a biological responseresulting from the binding to or cleavage of aggrecan by aggrecanases ofthe invention.

The term “deletion” as used herein is the removal of at least one aminoacid from the full-length amino acid sequence of an aggrecanase. Theterm deletion also refers to removal of nucleotides from a nucleic acidsequence encoding an aggrecanase, thereby resulting in a nucleic acidthat encodes a truncated protein.

A deletion in a nucleic acid molecule encoding an aggrecanase or anaggrecanase protein can be made anywhere in the nucleic acid molecule orthe protein as desirable. For example, a deletion may occur anywherewithin the protein; for example, N-terminus, C-terminus or any otherpart of an aggrecanase protein, and can include removal of at least oneamino acid; for example, from about 1 to about 5 amino acids, from about5 to about 10 amino acids, from about 10 to about 20 amino acids, fromabout 20 to about 30 amino acids, from about 30 to about 50 amino acids,from about 50 to about 100 amino acids, from about 100 to about 150amino acids, from about 150 to about 200 amino acids, from about 200 toabout 250 amino acids, from about 250 to about 300 amino acids, fromabout 300 to about 350 amino acids, from about 350 to about 400 aminoacids, from about 400 to about 450 amino acids, from about 450 to about500 amino acids, or greater than 500 amino acids.

Deletions can also be made in nucleic acid molecules that encodeaggrecanases of the invention; for example, deletions can be made in theregion of a nucleic acid that encodes for a TSP domain of anaggrecanase. Such deletions typically encompass the 3′ region of anucleic acid molecule encoding an aggrecanase of the invention. However,it is contemplated that deletions can be made anywhere in a nucleic acidexpressing an aggrecanase molecule. One skilled in the art can testtruncated aggrecanases of the invention for activity in one of manyassays disclosed.

An amino acid deletion according to the invention comprises the removalof at least one amino acid from the N-terminus of an aggrecanaseprotein. In another embodiment, a deletion comprises removal of at leastone amino acid from the C-terminus of an aggrecanase protein. In yetanother embodiment, a deletion comprises removal of amino acids from aregion lying between N-terminal end and C-terminal end of an aggrecanasemolecule. In one embodiment, such a deletion involves removal of anentire domain of an aggrecanase of the invention. For example,aggrecanases of the invention comprising a deletion includeaggrecanase-1 molecules that have one TSP domain deleted oraggrecanase-1 molecules that have two TSP domains deleted, oraggrecanase-2 molecules that have one TSP domain deleted, oraggrecanase-2 molecules that have two TSP domains deleted. Aggrecanasesaccording to the invention may comprise deletion of all TSP domainswithin an aggrecanase protein. It is contemplated that other domainswithin aggrecanase molecules may also be deleted to generatebiologically active truncated aggrecanases that are more stable than thefull-length counterpart.

The term “fragment” as used herein, refers to a portion of anaggrecanase protein of the invention, for example, a portion of aminoacid sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, 11, 12, and 13.In one embodiment, a fragment of a protein refers to an amino acidsequence that has aggrecanase activity in one of many assays provided.The term “fragment” also includes nucleotide sequences that are longenough to encode peptides that exhibit aggrecanase activity. However,fragments of a nucleotide sequence may or may not encode proteinfragments that retain aggrecanase biological activity. Protein andnucleic acid fragments of the invention include portions of othernucleic acid molecules or proteins that are substantially identical toat least one portion of the nucleotide sequences set forth in SEQ IDNOs: 1, 3, 5, 7, 9, 31, 32, or 33, or at least one portion of amino acidsequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 11, 12, or 13,respectively. Fragments of nucleic acid sequences may range, forexample, from at least about 20 nucleotides, from at least about 50nucleotides, from at least about 100 nucleotides, from at least about150 nucleotides, from at least about 200 nucleotides, from at leastabout 250 nucleotides, from at least about 300 nucleotides, from atleast about 400 nucleotides, from at least about 500 nucleotides, fromat least about 600 nucleotides, from at least about 700 nucleotides,from at least about 800 nucleotides, up to the entire length of thenucleic acid sequence set forth in SEQ ID NO: 1. In one embodiment,nucleic acid fragments encode peptides that have aggrecanase activity.Protein fragments may range, for example, from at least about 5 aminoacids, from at least about 10 amino acids, from at least about 20 aminoacids, from about 30 amino acids, from about 40 amino acids, from about50 amino acids, from at least about 100 amino acids, from at least about150 amino acids, from at least about 200 amino acids, from at leastabout 250 amino acids, from at least about 300 amino acids, from atleast about 350 amino acids, from at least about 400 amino acids, up tothe entire length of the amino acid sequence set forth in SEQ ID NO: 2.Fragments of nucleic acids of the invention can arise from 3′ portions,5′ portions or any other part of a nucleic acid sequence. Similarly,protein fragments can arise from N-terminus portion, C-terminus portionor any other part of a protein. In one embodiment, fragments of proteinsretain aggrecanase activity. In another embodiment, protein fragments ofthe invention arise from a portion of a protein that has aggrecanaseactivity.

The term “effective amount” refers to a dosage or an amount of acomposition of at least one aggrecanase inhibitor or antibody of theinvention that is sufficient to treat a patient.

The term “inhibit” or “inhibition” of aggrecanase or aggrecanaseactivity refers to a reduction, inhibition of otherwise diminution of atleast one activity of aggrecanase due to binding of an inhibitor to theaggrecanase or aggrecan. The reduction, inhibition, or diminution ofbinding can be measured by one of many assays provided. Inhibition ofaggrecanase activity does not necessarily indicate a complete negationof aggrecanase activity. A reduction in activity can be, for example, atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In oneembodiment, inhibition is measured by a reduction in the detection ofcleavage products of aggrecan. Inhibitors of the present inventioninclude, but are not limited to, antibodies, proteins, peptides, andchemical compounds (often referred to as small molecules).

The term “isolated” describes a nucleic acid molecule or polypeptidemolecule that is substantially free of its natural environment. Forinstance, an isolated protein is substantially free of cellular materialor other contaminating proteins from the cell or tissue source fromwhich it is derived. The term “isolated” also refers to an aggrecanaseprotein according to the invention which is free from association withother proteases and retains aggrecanase proteolytic activity. Inaddition, the term “isolated” refers to nucleic acid molecules thatencode aggrecanases of the invention and are free from other cellularmaterial and contaminants.

The term “neoepitope antibody” refers to an antibody that specificallyrecognizes a new N- or C-terminal amino acid sequence generated byproteolytic cleavage but which does not bind to such an epitope on theintact (uncleaved) substrate.

The term “operative association” with an expression control sequencegenerally refers to the presence of a specific nucleotide sequence orsequences that control or affect transcription rate or efficiency of anucleotide molecule linked to the sequence. For example, a promotersequence that is located proximally to the 5′ end of an aggrecanasecoding nucleotide sequence may be in operative association with theaggrecanase encoding nucleotide sequence. Expression control sequencesinclude, but are not limited to, for example, promoters, enhancers, andother expression control sequences, or any combination of such elements,either 5′ or 3′ to an aggrecanase encoding nucleotide sequence in orderto control its expression. Not all of these elements are required,however. A skilled artisan can select the appropriate expression controlsequences, for example, depending on desired expression levels for theaggrecanases of the invention.

The term “specific binding” of an antibody means that the antibody bindsto at least one aggrecanase molecule of the present invention and theantibody will not show any significant binding to molecules other thanat least one novel aggrecanase molecule. The term is also applicablewhere, e.g., an antigen binding domain of an antibody is specific for aparticular epitope, which is represented on a number of antigens, andthe specific binding member (the antibody) carrying the antigen bindingdomain will be able to bind to the various antigens carrying theepitope. Therefore, it is contemplated that an antibody of the inventionwill bind to an epitope on multiple novel aggrecanase proteins.Typically, the binding is considered specific when the affinity constantK_(a) is higher than 10⁸ M⁻¹. An antibody is said to “specifically bind”to an antigen if, under appropriately selected conditions, such bindingis not substantially inhibited, while at the same time non-specificbinding is inhibited. The conditions are usually defined in terms ofconcentration of antibodies, ionic strength of the solution,temperature, time allowed for binding, concentration of additionalmolecules associated with the binding reaction (e.g., serum albumin,milk casein), etc. Such conditions are well known in the art, and askilled artisan using routine techniques can select appropriateconditions.

The term “stability” as used herein, generally refers to a decrease inthe rate of degradation of a protein, thereby increasing its half-life,solubility, and/or expression levels. Several factors affect proteinstability in vitro and in vivo, for example, pH, salt concentration,temperature, protein degradation, for example by proteases, metal ions,autocatalysis of proteins, hydrophobicity etc. In one embodiment, theinvention includes truncated aggrecanases that are more stable thantheir full-length counterparts. In another embodiment, the inventionincludes aggrecanase active-site mutants that are more stable than theirwild-type counterparts. Conditions that make a protein more stablegenerally include conditions that keep the protein in a foldedconformation for longer than normal, thereby preserving its biologicalactivity for a longer period of time. An increase in stability of aprotein generally increases its half-life and expression levels, therebymaking it possible to purify the protein in large amounts fortherapeutic purposes and for development of inhibitors.

The term “highly stringent” or “high stringency” describes conditionsfor hybridization and washing used for determining nucleic acid-nucleicacid interactions. Nucleic acid hybridization will be affected by suchconditions as salt concentration, temperature, or organic solvents, inaddition to the base composition, length of the complementary strands,and the number of nucleotide base mismatches between the hybridizingnucleic acids, as will be readily appreciated by those skilled in theart. The stringency conditions are dependent on the length of thenucleic acid and the base composition of the nucleic acid and can bedetermined by techniques well known in the art. Generally, stringencycan be altered or controlled by, for example, manipulating temperatureand salt concentration during hybridization and washing. For example, acombination of high temperature and low salt concentration increasesstringency. Such conditions are known to those skilled in the art andcan be found in, for example, Current Protocols in Molecular Biology,John Wiley & Sons, N.Y., 6.3.1–6.3.6, (1989). Both aqueous andnonaqueous conditions as described in the art can be used. One exampleof highly stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by atleast one wash in 0.2×SSC, 0.1% SDS at 50° C. A second example of highlystringent hybridization conditions is hybridization in 6×SSC at about45° C., followed by at least one wash in 0.2×SSC, 0.1% SDS at 55° C.Another example of highly stringent hybridization conditions ishybridization in 6×SSC at about 45° C., followed by at least one wash in0.2×SSC, 0.1% SDS at 60° C. A further example of highly stringenthybridization conditions is hybridization in 6×SSC at about 45° C.,followed by at least one wash in 0.2×SSC, 0.1% SDS at 65° C. Highlystringent conditions include hybridization in 0.5M sodium phosphate, 7%SDS at 65° C., followed by at least one wash at 0.2×SSC, 1% SDS at 65°C.

The phrase “moderately stringent” or “moderate stringency” hybridizationrefers to conditions that permit a nucleic acid to bind a complementarynucleic acid that has at least about 60%, at least about 75%, or atleast about 85%, identity to the nucleic acid, with greater than about90% identity to the nucleic acid especially preferred. Moderatelystringent conditions comprise but are not limited to, for example,hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C. (see,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989).

The phrase “substantially identical” or “substantially similar” meansthat the relevant amino acid or nucleotide sequence will be identical toor have insubstantial differences (through conserved amino acidsubstitutions) in comparison to the sequences which are disclosed.Nucleotide and polypeptides of the invention include, for example, thosethat are at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%identical in sequence to nucleic acid molecules and polypeptidesdisclosed.

For polypeptides, at least 20, 30, 50, 100, or more amino acids will becompared between the original polypeptide and the variant polypeptidethat is substantially identical to the original. For nucleic acids, atleast 50, 100, 150, 300, or more nucleotides will be compared betweenthe original nucleic acid and the variant nucleic acid that issubstantially identical to the original. Thus, a variant could besubstantially identical in a region or regions, but divergent in others,while still meeting the definition of “substantially identical.” Percentidentity between two sequences is determined by standard alignmentalgorithms such as, for example, Basic Local Alignment Tool (BLAST)described in Altschul et al., J. Mol. Biol., 215:403–410 (1990), thealgorithm of Needleman and Wunsch, J. Mol. Biol., 48:444–453 (1970), orthe algorithm of Meyers and Miller, Comput. Appl. Biosci., 4:11–17(1988).

The term “treating” or “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentmay include individuals already having a particular medical disorder aswell as those who may ultimately acquire the disorder (i.e., thoseneeding preventative measures). Treatment may regulate aggrecanaseactivity or the level of aggrecanase to prevent or ameliorate clinicalsymptoms of at least one disease. The inhibitors and/or antibodies mayfunction by, for example, preventing the interaction or binding ofaggrecanase to aggrecan, or by reducing or inhibiting aggrecanaseactivity.

The term “truncated” as used herein, refers to nucleotides encodingaggrecanases of the invention that are missing at least one nucleotide,or aggrecanases of the invention that are missing at least one aminoacid, which are found in a full-length aggrecanase nucleic acid moleculeor amino acid sequence, respectively. Truncated proteins of theinvention are generally shorter in length compared with theirfull-length counterparts, usually due to a deletion of at least onenucleotide in a nucleic acid sequence encoding a truncated protein or inat least one amino acid of the corresponding full-length protein.

In one embodiment, for example, truncated aggrecanases of the inventioninclude truncated aggrecanase-1 proteins comprising an amino acidsequence set forth in SEQ ID NO: 12 comprising deletion of amino acid#576 through the C-terminal end of the full-length aggrecanase-1protein, set forth in SEQ ID NO: 11; and SEQ ID NO: 13 comprisingdeletion of amino acid #521 through the C-terminal end of thefull-length aggrecanase-1 protein, set forth in SEQ ID NO: 11. Inanother embodiment, truncated aggrecanases of the invention compriseaggrecanase-2 truncated proteins; for example, as set forth in SEQ IDNO: 4 comprising deletion of amino acid #754 through the C-terminal endof the full-length aggrecanase-2 protein, set forth in SEQ ID NO: 2; SEQID NO: 6 comprising deletion of amino acid #753 through the C-terminalend of the full-length aggrecanase-2 protein, set forth in SEQ ID NO: 2;SEQ ID NO: 8 comprising deletion of amino acid #629 through theC-terminal end of the full-length aggrecanase-2 protein, set forth inSEQ ID NO: 2; and SEQ ID NO: 10 comprising deletion of amino acid #568through the C-terminal end of the full-length aggrecanase-2 protein, setforth in SEQ ID NO: 2. Such a deletion can be made either in a nucleicacid that encodes an aggrecanase or be made in the protein itselfsubsequent to the formation of the protein. Accordingly, the term“truncated” as used herein refers to nucleic acids that encodeaggrecanases that are truncated as well as truncated aggrecanasesthemselves. Nucleic acids encoding for truncated proteins of theinvention are depicted in SEQ ID NOs: 3, 5, 7, 9, 11, 32, and 33.Truncated proteins of the invention can be expressed as fusion proteins.

Truncated aggrecanases of the invention have greater stability than thecorresponding full-length aggrecanase molecule. Truncated aggrecanasesof the invention can also be expressed at higher levels both in vivo andin vitro than the corresponding full-length aggrecanase proteins.Truncated aggrecanases of the invention are biologically active and mayinclude other alterations, such as amino acid substitutions,modifications, or deletions in other parts of the protein.

The term “variant” refers to nucleotide and amino acid sequences thatare substantially identical or similar to the nucleotide and amino acidsequences provided, respectively. Variants can be naturally occurring,for example, naturally occurring human and non-human nucleotidesequences that encode aggrecanase or aggrecanase-like proteins, or begenerated artificially. Examples of variants are aggrecanases resultingfrom alternative splicing of the aggrecanase mRNA, including both 3′ and5′ spliced variants of the aggrecanases of the invention, pointmutations and other mutations, or proteolytic cleavage of theaggrecanase protein. Variants of aggrecanases of the invention includenucleic acid molecules or fragments thereof and amino acid sequences andfragments thereof, that are substantially identical or similar to othernucleic acids (or their complementary strands when they are optimallyaligned (with appropriate insertions or deletions)) or amino acidsequences respectively. In one embodiment, there is at least about 50%identity, at least about 55% identity, at least about 60% identity, atleast about 65% identity, at least about 70% identity, at least about75% identity, at least about 80% identity, at least about 85% identity,at least about 90%, at least about 92% identity, at least about 93%identity, at least about 94% identity, at least about 95% identity, atleast about 96% identity, at least about 97% identity, at least about98% identity, or at least about 99% identity between a nucleic acidmolecule or protein of the invention and another nucleic acid moleculeor protein respectively, when optimally aligned. Variants, as defined,include both naturally occurring nucleic acid sequences as well asequivalent degenerative codon sequences of the aggrecanases of theinvention. Additionally, variants include proteins or polypeptides thatexhibit aggrecanase activity, as defined.

To assist in the identification of the sequences listed in thespecification and figures, the following table (Table 1) is provided,which lists the SEQ ID NOs, the figure location, and a brief descriptionof each sequence.

TABLE 1 SEQUENCES FIGURES DESCRIPTION SEQ ID NO: 1 FIGS. 1A–1Cfull-length nucleotide sequence of ADAMTS-5 (aggrecanase-2) SEQ ID NO: 2FIG. 2 full-length a.a. sequence of ADAMTS-5 encoded by SEQ ID NO: 1 SEQID NO: 3 FIG. 3 a nucleotide sequence from nucleotide #123 to nucleotide#2382 of SEQ ID NO: 1 SEQ ID NO: 4 FIG. 4 a.a. sequence of a truncatedaggrecanase-2 molecule encoded by SEQ ID NO: 3 SEQ ID NO: 5 FIG. 5 anucleotide sequence from nucleotide #123 to nucleotide #2379 of SEQ IDNO: 1 SEQ ID NO: 6 FIG. 6 a.a. sequence of a truncated aggrecanase-2molecule encoded by SEQ ID NO: 5 SEQ ID NO: 7 FIG. 7 a nucleotidesequence from nucleotide #123 to nucleotide #2007 of SEQ ID NO: 1 SEQ IDNO: 8 FIG. 8 a.a. sequence of a truncated aggrecanase-2 molecule encodedby SEQ ID NO: 7 SEQ ID NO: 9 FIG. 9 a nucleotide sequence fromnucleotide #123 to nucleotide #1824 of SEQ ID NO: 1 SEQ ID NO: 10 FIG.10 a.a. sequence of a truncated aggrecanase-2 molecule encoded by SEQ IDNO: 9 SEQ ID NO: 11 FIG. 11 a.a. sequence of full- length aggrecanase-1protein encoded by SEQ ID NO: 31 SEQ ID NO: 12 FIG. 12 a.a. sequence oftruncated aggrecanase-1 protein including a.a #1 (Met) through a.a #575(Pro) encoded by SEQ ID NO: 32 SEQ ID NO: 13 FIG. 13 a.a. sequence oftruncated aggrecanase-1 protein including a.a. #1 (Met) through a.a.#520 (Ala) encoded by SEQ ID NO: 33 SEQ ID NO: 14 FIG. 14 a nucleotidesequence of a recombinant truncated aggrecanase-2 protein with a peptidelinker and a streptavidin tag SEQ ID NO: 15 FIG. 15 a.a. sequence of arecombinant aggrecanase-2 protein encoded by SEQ ID NO: 14. SEQ ID NO:16 Primer SEQ ID NO: 17 Primer SEQ ID NO: 18 Primer SEQ ID NO: 19 PrimerSEQ ID NO: 20 oligonucleotide SEQ ID NO: 21 oligonucleotide SEQ ID NO:22 peptide linker SEQ ID NO: 23 FIG. 18 streptavidin - tag SEQ ID NO: 24oligonucleotide SEQ ID NO: 25 oligonucleotide SEQ ID NO: 26 catalyticmotif SEQ ID NO: 27 Nucleotide insert SEQ ID NO: 28 insert containingXho1 site SEQ ID NO: 29 68 bp adapter SEQ ID NO: 30 FIG. 21aggrecanase-2 protein with an E-to-Q mutation at a.a. position 411 SEQID NO: 31 FIGS. 22A and 22B full-length nucleotide sequence ofaggrecanase-1 SEQ ID NO: 32 FIG. 23 Nucleotide sequence of a truncatedaggrecanase-1 SEQ ID NO: 33 FIG. 24 Nucleotide sequence of a truncatedaggrecanase-1 a.a. = amino acidII. Novel Aggrecanase Molecules

In one embodiment, the nucleotide sequence of a truncated aggrecanase-2of the invention is set forth in SEQ ID NO: 3, wherein the nucleic acidsequence includes nucleotide #123 through nucleotide #2382 of thefull-length nucleic acid sequence of aggrecanase-2, set forth in SEQ IDNO: 1. In another embodiment the nucleotide sequence of a truncatedaggrecanase-2 is set forth in FIG. 5 from nucleotide #1 throughnucleotide #2256. In another embodiment the nucleotide sequence of atruncated aggrecanase-2 of the invention comprises nucleotide #1 through#1884 set forth in FIG. 7, which comprises deletion of a nucleotidesequence encoding a TSP domain. In yet another embodiment, the nucleicacid sequence of a truncated aggrecanase-2 protein of the inventioncomprises nucleotide #1 through nucleotide #1701 which encodes anaggrecanase-2 protein having both TSP domains deleted. The inventionfurther includes naturally occurring nucleic acid sequences andfragments and variants thereof, as well as those that are artificiallygenerated and equivalent degenerative codon sequences of the sequencedescribed above as set forth in FIGS. 3, 5, 7, and 9, as well asfragments thereof which encode polypeptides that exhibit aggrecanaseactivity.

In another embodiment, the nucleotide sequences of the invention includenucleic acid sequences that encode truncated aggrecanase-1 molecules.For example, a truncated aggrecanase-1 protein, as set forth in FIG. 12,can be encoded by the nucleic acid sequence set forth in FIG. 23 (SEQ IDNO: 32). Yet another truncated aggrecanase-1 protein of the invention isset forth in FIG. 13, which includes deletion of a TSP domain. A nucleicacid sequence that encodes such a truncated aggrecanase-1 protein is setforth in FIG. 24 (SEQ ID NO: 33).

The amino acid sequence of an isolated truncated aggrecanase-2 moleculeis set forth in FIG. 4 from amino acid #1 (Met) through #753 (Glu). Theinvention further features amino acid sequences encoded by nucleotidesequences of FIGS. 5, 7, and 9, which encode truncated aggrecanase-2proteins, amino acid sequences of which are set forth in: FIG. 6including amino acid #1 (Met) through #752 (Pro); FIG. 8 including aminoacid #1 (Met) through amino acid #628 (Phe); and FIG. 10 including aminoacid #1 (Met) through amino acid #567 (His). The invention furtherincludes amino acid sequences for truncated aggrecanase-1 molecules. Theamino acid sequence for a truncated aggrecanase-1 molecule is set forthin FIG. 23, which may be encoded by the nucleotide sequence of FIG. 12.Yet another truncated aggrecanase-1 molecule of the invention isfeatured in FIG. 13, which may be encoded by the nucleic acid sequenceof FIG. 24. The invention further includes fragments and variants of theamino acid sequences of polypeptides that exhibit aggrecanase activity.

A human aggrecanase protein of the invention or a fragment thereof maybe produced by culturing a cell transformed with a DNA sequence of FIG.3 from nucleotide #1 through #2259 and recovering and purifying from theculture medium a protein characterized by the amino acid sequence setforth in FIG. 4 from amino acid #1 (Met) through #753 (Glu)substantially free from other proteinaceous materials with which it isco-produced.

In another embodiment, the aggrecanase protein of the invention may beproduced by culturing a cell transformed with the DNA sequence of FIG. 5from nucleotide #1 through nucleotide #2256 and recovering and purifyingthe aggrecanase protein comprising an amino acid sequence of FIG. 6 fromamino acid #1 (Met) through amino acid #752 (Pro). In anotherembodiment, the aggrecanase protein of the invention may be produced byculturing a cell transformed with the DNA sequence of FIG. 5 fromnucleotide #1 to nucleotide #2256 with an additional GAA following CCTand recovering and purifying the aggrecanase protein comprising an aminoacid sequence of FIG. 4 from amino acid #1 (Met) through amino acid #753(Glu). In yet another embodiment aggrecanase proteins of the presentinvention may be produced by culturing a cell transformed with the DNAsequence of FIGS. 1A–1C (SEQ ID NO: 1) and recovering and purifying atruncated aggrecanase protein from the culture medium comprising aminoacid #1 through amino acid #752 (SEQ ID NO: 6), or amino acid #1 throughamino acid #753 (SEQ ID NO: 4), produced due to cleavage of thefull-length aggrecanase-2 protein set forth in SEQ ID NO: 2.

In a further embodiment, a protein recovered from a cell culture mediumincludes amino acids #1 through #628 (SEQ ID NO: 8); amino acids #1though 567 (SEQ ID NO: 10); amino acids #1 through #575 (SEQ ID NO: 12)or amino acids #1 through #520 (SEQ ID NO: 13). Purified expressedproteins are substantially free from other proteinaceous materials withwhich they are co-produced, as well as from other contaminants. Arecovered purified protein is contemplated to exhibit proteolyticaggrecanase activity by cleaving aggrecan. Thus, proteins of theinvention may be further characterized by their ability to demonstrateaggrecan proteolytic activity in an assay which determines the presenceof an aggrecan-degrading molecule. These assays or the developmentthereof is within the knowledge of one skilled in the art. Such assaysmay involve contacting an aggrecan substrate with the aggrecanasemolecule and monitoring the production of aggrecan fragments (see, forexample, Hughes et al., Biochem J 305:799–804 (1995); Mercuri et al., J.Bio Chem. 274:32387–32395 (1999)).

For production in mammalian cells, the DNA sequence further comprises aDNA sequence encoding a suitable propeptide 5′ to and linked in frame tothe nucleotide sequence encoding an aggrecanase enzyme. Aggrecanaseproteins of the invention recovered from a culture medium are purifiedby isolating them from other proteinaceous materials with which they areco-produced and from other contaminants present. The isolated andpurified proteins may be characterized by the ability to cleave aggrecansubstrate.

Aggrecanase proteins provided herein also include proteins encoded bythe sequences similar to those of FIG. 3 from nucleotide #1 through#2259; FIG. 5 from nucleotide #1 through nucleotide #2256; FIG. 7 fromnucleotide #1 through nucleotide #1884; FIG. 9 from nucleotide #1through nucleotide #1701; FIG. 23 from nucleotide #1 through nucleotide#1725; and FIG. 24 from nucleotide #1 through nucleotide #1560, but intowhich modifications or deletions are naturally provided (e.g., allelicvariations in the nucleotide sequence which may result in amino acidchanges in the polypeptide) or deliberately engineered. For example,synthetic polypeptides may wholly or partially duplicate continuoussequences of the amino acid residues of FIG. 4 from amino acid #1 (Met)through amino acid #753 (Glu); FIG. 6 from amino acid #1 (Met) through#752 (Pro); FIG. 8 from amino acid #1 (Met) through amino acid #628(Phe); FIG. 10 from amino acid #1 (Met) through amino acid #567 (His);FIG. 12 from amino acid #1 (Met) through amino acid #575 (Pro); or FIG.13 from amino acid #1 (Met) through amino acid #520 (Ala). Thesesequences, by virtue of sharing primary, secondary, or tertiarystructural and conformational characteristics with aggrecanasemolecules, may possess biological properties in common therewith.

It is known, for example, that numerous conservative amino acidsubstitutions are possible without significantly modifying the structureand conformation of a protein, thus maintaining the biologicalproperties as well. For example, it is recognized that conservativeamino acid substitutions may be made among amino acids with basic sidechains, such as lysine (Lys or K), arginine (Arg or R) and histidine(His or H); amino acids with acidic side chains, such as aspartic acid(Asp or D) and glutamic acid (Glu or E); amino acids with unchargedpolar side chains, such as asparagine (Asn or N), glutamine (Gln or Q),serine (Ser or S), threonine (Thr or T), and tyrosine (Tyr or Y); andamino acids with nonpolar side chains, such as alanine (Ala or A),glycine (Gly or G), valine (Val or V), leucine (Leu or L), isoleucine(lie or I), proline (Pro or P), phenylalanine (Phe or F), methionine(Met or M), tryptophan (Trp or W), and cysteine (Cys or C). Thus, thesemodifications and deletions of the native aggrecanase may be employed asbiologically active substitutes for naturally occurring aggrecanase andin the development of inhibitors of other polypeptides in therapeuticprocesses. It can be readily determined whether a given variant ofaggrecanase maintains the biological activity of aggrecanase bysubjecting both aggrecanase and the variant of aggrecanase, as well asinhibitors thereof, to the assays described in the examples.

The invention also includes aggrecanase molecules comprising amino acidsubstitutions that increase the stability of the aggrecanase molecules.For example, the amino acid sequence of an aggrecanase-2 molecule withan E-to-Q mutation at position 411 of the protein is provided in SEQ IDNO: 30. FIG. 17 further provides schematic representations of amino acidmutations E-to-Q within the catalytic domains of truncated aggrecanase-1molecules of FIGS. 12 and 13. Although the E-to-Q mutation is in theactive site of the molecule and serves to prevent degradation ofaggrecanases of the invention, and to thereby increase stability andhalf-life of aggrecanases, it is contemplated that amino acid mutationscan be made in other regions of the protein that increase stability ofaggrecanase molecules.

In one embodiment, active site mutations are introduced intoaggrecanases to intentionally block the catalytic activity of theenzyme. This is especially useful for the purposes of crystallizationand structural determination of aggrecanases and subsequently toidentify and develop inhibitors of aggrecanases. Increased stability oftruncated or active-site mutant aggrecanases of the invention makes itpossible to purify and isolate large amounts of aggrecanase moleculesfor subsequent use in the development of inhibitors for treatment ofdiseases. The E-to-Q mutation makes the aggrecanases biologicallyinactive, thereby enabling purification of inactive protein in largeamounts for use in gene therapy in patients and also for development ofinhibitors.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important amino acid residues ofthe proteins or polypeptides of the invention or to increase or decreasethe activity of the aggrecanases of the invention described. Exemplaryamino acid substitutions are set forth in Table 2.

TABLE 2 Amino Acid Substitutions More Original Exemplary ConservativeResidues Substitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R)Lys, Gln, Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala SerGln (Q) Asn Asn Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile(I) Leu, Val, Met, Ala, Phe, Norleucine Leu Leu (L) Norleucine, Ile,Val, Met, Ala, Phe Ile Lys (K) Arg, 1,4 Diamino-butyric Acid, Gln, AsnArg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro(P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, PheTyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Ala,Norleucine Leu

In certain embodiments, conservative amino acid substitutions alsoencompass non-naturally occurring amino acid residues which aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems.

Other specific mutations of the sequences of aggrecanase proteinsdescribed herein involve modifications of glycosylation sites. Thesemodifications may involve O-linked or N-linked glycosylation sites. Forinstance, the absence of glycosylation or only partial glycosylationresults from amino acid substitution or deletion at asparagine-linkedglycosylation recognition sites. The asparagine-linked glycosylationrecognition sites comprise tripeptide sequences which are specificallyrecognized by appropriate cellular glycosylation enzymes. Thesetripeptide sequences are either asparagine-X-threonine orasparagine-X-serine, where X is usually any amino acid. A variety ofamino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Additionally, bacterial expressionof aggrecanase-related protein will also result in production of anon-glycosylated protein, even if the glycosylation sites are leftunmodified.

III. Aggrecanase Nucleotide Sequences

Nucleic acids within the scope of the invention include isolated DNA andRNA sequences that hybridize to the native aggrecanase DNA sequencesdisclosed under conditions of moderate to high stringency. Conditions ofhigh stringency generally refer to hybridization and washing conditionsthat employ higher temperatures and lower salt concentrations.Additionally, inclusion of formamide also increases stringency. Forexample, hybridization conditions at 60–65° C. in the absence offormamide or at 42° C. with 50% formamide, are both high-stringencyconditions.

Further included in the present invention are DNA sequences whichhybridize under high to moderate stringent conditions with the DNAsequence of SEQ ID NOs: 3, 5, 7, 9, 32, or 33 and encode a truncatedaggrecanase protein having the ability to cleave aggrecan. In oneembodiment, DNA sequences include those which hybridize under highstringent conditions (see Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, pgs. 387–389 (1982)).Such stringent conditions comprise, for example, 0.1×SSC/0.1% SDS at 65°C. DNA sequences identified by hybridization include, for example, DNAsequences that encode a protein which is at least about 80% identical,at least about 90% identical, or at least about 95% identical to thesequence of the proteins of the invention, for example, amino acidsequences set forth in SEQ ID NOs: 4, 6, 8, 10, 12, and 13. DNAs thatare equivalents to the DNAs of SEQ ID NOs: 3, 5, 7, 9, 32, or 33 willalso hybridize under moderately stringent conditions to the DNA sequenceencoding the peptide sequence of SEQ ID NO: 4, 6, 8, 10, 12, or 13,respectively. It is understood, however, that under certain conditions,nucleic acid molecules that encode truncated proteins will hybridize tonucleic acid molecules encoding full-length proteins.

Conditions of moderate stringency are known in the art and are definedby Sambrook et al., Molecular Cloning: A Laboratory Manual, Vol. 1, ColdSpring Harbor Press (2^(nd) ed. 1989). In one embodiment, for example,conditions of moderate stringency include use of a prewashing solutionof 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), and hybridization conditionsof about 55° C.–60° C. temperature and washing overnight in 5×SSCovernight at about 55° C. The skilled artisan will recognize that theconditions may be adjusted as necessary according to factors such as thelength and composition of the nucleic acid sequences.

Still a further aspect of the invention are DNA sequences encodingtruncated aggrecanases having aggrecanase proteolytic activity or otheryet undisclosed or undiscovered activities of aggrecanases.

In yet another embodiment, nucleic acids of the invention includenucleic acid molecules that encode aggrecanases comprising mutationsthat lead to increase in stability of such molecules.

Finally, alleic or other variations of the sequences of the invention,for example, SEQ ID NOs: 3, 5, 7, 9, 32, and 33, encoding amino acidsequences set forth in SEQ ID NOs: 4, 6, 8, 10, 12, and 13 respectively,or peptide sequence variants of aggrecanases of the invention that haveaggrecanase activity, are also included in the present invention.Additionally, the present invention includes fragments of the DNAsequences of the invention and variants of such sequences that encode aprotein with aggrecanase activity.

Similarly, DNA sequences that code for aggrecanase proteins but whichdiffer in codon sequence due to the degeneracies of the genetic code orallelic variations (naturally occurring base changes in the speciespopulation which may or may not result in an amino acid change) alsoencode novel aggrecanases described herein. Variations in the DNAsequences of FIGS. 1, 3, 5, 7, 9, 31, 32, and 33, which are caused bypoint mutations or by induced modifications (including insertion,deletion, and substitution) to enhance the activity, half-life, orproduction of the polypeptides encoded are also encompassed in theinvention.

Another aspect of the present invention includes vectors for use in amethod of expression of these novel aggrecanase polypeptides.Preferably, vectors of the invention contain a DNA sequence describedabove which encodes a truncated aggrecanase or an active site mutantaggrecanase of the invention. Additionally, vectors contain appropriateexpression control sequences permitting expression of aggrecanaseprotein sequences of the invention. Alternatively, vectors incorporatingmodified sequences as described above are also embodiments of thepresent invention. Additionally, the sequence of FIG. 3 from nucleotide#1 through #2259, or FIG. 5 from nucleotide #1 through #2256, or FIG. 7from nucleotide #1 through nucleotide #1884, or FIG. 9 from nucleotide#1 through nucleotide #1701, or FIG. 23 from nucleotide #1 throughnucleotide #1726, or FIG. 24 from nucleotide #1 through nucleotide#1561, or other sequences encoding aggrecanase proteins could bemanipulated to express composite aggrecanase molecules. Thus, thepresent invention includes chimeric DNA molecules encoding anaggrecanase protein comprising a fragment from nucleotide sequences setforth in SEQ ID NOs: 3, 5, 7, 9, 32, and 33 linked in correct readingframe to a DNA sequence encoding another aggrecanase polypeptide. A DNAmolecule as set forth in SEQ ID NOs: 3, 5, 7, 9, 32, or 33, or a variantor fragment thereof, may be linked either 5′ or 3′ to a DNA moleculeencoding another aggrecanase.

IV. Production of Aggrecanase Proteins

Another aspect of the present invention provides methods for producingaggrecanase proteins. In one embodiment, a method of the presentinvention involves culturing a suitable cell line, which has beentransformed with a DNA sequence encoding an aggrecanase protein of theinvention under the control of known regulatory sequences. Thetransformed host cells are cultured and the aggrecanase proteins arerecovered and purified from the culture medium. The purified proteinsare substantially free from other proteins with which they areco-produced, as well as from other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). The selection of suitable mammalian hostcells and methods for transformation, culture, amplification, screening,product production, and purification are known in the art. See, e.g.,Gething and Sambrook, Nature, 293:620–625 (1981), Kaufman et al., Mol.Cell. Biol., 5(7):1750–1759 (1985), or Howley et al., U.S. Pat. No.4,419,446. Another suitable mammalian cell line, which is described inthe accompanying examples, is the monkey COS-1 cell line. The mammaliancell line CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli, and the like may also be employed in this method. Forexpression of aggrecanase proteins of the invention in bacterial cells,DNA encoding the propeptide of an aggrecanase is generally notnecessary.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of polypeptides of the presentinvention. Additionally, where desired, insect cells may be utilized ashost cells in the method of the present invention. See, e.g. Miller etal., Genetic Engineering, 8:277–298, Plenum Press (1986) and referencescited therein.

Various vectors disclosed may be employed in the method of transformingcell lines and usually contain selected regulatory sequences inoperative association with the DNA coding sequences of the inventionwhich are capable of directing the replication and expression thereof inselected host cells. Regulatory sequences for such vectors are known tothose skilled in the art and may be selected, depending upon the hostcells. Such selection is routine and does not form part of the presentinvention.

One skilled in the art can construct mammalian vectors by employing anucleic acid sequence disclosed, for example, SEQ ID NOs: 1, 3, 5, 7, 9,31, 32, and 33.

Truncated aggrecanase proteins of the invention can also be expressed asfusion proteins including the protein sequence. For example, thesequence set forth in SEQ ID NO: 4, 6, 8, 10, 12, or 13, or a fragmentor a variant thereof, and, for example, a tag (i.e., a second protein orat least one amino acid) from about 2 to 50 amino acids, or from about50 to about 100 amino acids, which are added to the amino terminus of,the carboxy terminus of, or any point within the amino acid sequence ofan aggrecanase protein, or a fragment or variant thereof. Typically,such amino acid tags are made to stabilize the resulting fusion proteinor to simplify purification of an expressed recombinant form of thecorresponding aggrecanase protein or a fragment or a variant of suchprotein, including, for example, a truncated form of an aggrecanaseprotein of the invention. Such tags are known in the art. Representativeexamples of such tags include sequences which encode a series ofhistidine residues, the epitope tag FLAG, the Herpes simplexglycoprotein D, beta-galactosidase, maltose binding protein,streptavidin tag, or glutathione S-transferase. In one embodiment, anucleic acid sequence encoding a tag is linked in frame to a nucleicacid sequence encoding an aggrecanase of the invention for subsequentproduction of a recombinant or fusion aggrecanase protein including thetag. Such recombinant or fusion aggrecanase proteins include, forexample, truncated aggrecanase-1 enzymes with a C-terminal FLAG tag, asshown in FIG. 17, and truncated aggrecanase-2 enzymes with a C-terminalstreptavidin tag, as shown in FIG. 18. A nucleic acid sequence encodinga truncated aggrecanase-2 protein comprising a streptavidin tag is setforth in FIG. 14 (SEQ ID NO: 14), which encodes a truncatedaggrecanase-2 fusion protein as set forth in FIG. 15.

Similarly, aggrecanases that contain amino acid mutations which lead toincreased stability, expression levels, and/or half-lives can also beproduced as fusion proteins using, for example, the amino acid sequenceset forth in SEQ ID NO: 30, or a fragment or variant thereof, and, forexample, a tag as discussed above. Examples of aggrecanase-1 and -2fusion proteins of the invention, including aggrecanases with amino acidmutations in their catalytic domain, are set forth in FIGS. 14, 15, 17,and 18.

V. Generation of Antibodies

Isolated proteins of the present inventions may be used to generateantibodies, either monoclonal or polyclonal, to aggrecanase and/or otheraggrecanase-related proteins, using methods of antibody production thatare generally known in the art. Thus, the present invention alsoincludes antibodies to aggrecanase or other related proteins. Theantibodies include both antibodies that block aggrecanase activity andantibodies that do not. The antibodies may be useful for detectionand/or purification of aggrecanase or related proteins, or forinhibiting or preventing the effects of aggrecanase. Aggrecanases of theinvention or portions thereof may be utilized to prepare antibodies thatspecifically bind to aggrecanase.

Antibodies can be made, for example, via traditional hybridomatechniques (Kohler and Milstein, Nature 256:495–497 (1975)), recombinantDNA methods (for example, U.S. Pat. No. 4,816,567), or phage displaytechniques using antibody libraries (Clackson et al., Nature 352:624–628 (1991); Marks et al., J. Mol. Biol. 222:581–597 (1991)). Forvarious antibody production techniques, see Antibodies: A LaboratoryManual, eds. Harlow et al., Cold Spring Harbor Laboratory (1988).

Antibodies of the invention may be used in the treatment of the diseasesdescribed below. Antibodies can also be used in the assays and methodsof detection described.

VI. Development of Inhibitors

Various conditions such as osteoarthritis are known to be characterizedby degradation of aggrecan. Therefore, truncated aggrecanases of theinvention and aggrecanases with mutations that lead to increasedstability and expression levels of aggrecanases, make it possible togenerate aggrecanase molecules in large amounts in order to developinhibitors to aggrecanases.

The invention therefore provides compositions comprising an aggrecanaseinhibitor. Inhibitors may be developed using an aggrecanase molecule ofthe invention in screening assays involving a mixture of aggrecansubstrate with an inhibitor of aggrecanase activity followed by exposureto aggrecan. Inhibitors can be screened using high throughput processes,such as by screening a library of inhibitors. Inhibitors can also bemade using three-dimensional structural analysis and/or computer aideddrug design. The method may entail determination of binding sites forinhibitors based on the three-dimensional structure of aggrecanase andaggrecan and developing molecules reactive with a binding site onaggrecanase or aggrecan. Candidate molecules are assayed for inhibitoryactivity. Additional standard methods for developing inhibitors ofaggrecanase molecules are known to those skilled in the art. An assayfor identification and development of aggrecanase inhibitors involves,for example, contacting a mixture of aggrecan and an inhibitor with anaggrecanase molecule followed by measurement of the degree ofaggrecanase inhibition, for instance, by detection and measurement ofaggrecan fragments produced by cleavage at an aggrecanase susceptiblesite. Inhibitors may be proteins, peptides, antibodies, or chemicalcompounds. In one embodiment inhibitors are peptide molecules that bindan active site on aggrecanase molecules. For example, active sitemutants of aggrecanase-1 and aggrecanase-2 molecules can be used for thedevelopment of peptide inhibitors. Aggrecanase-1 molecules that comprisean E-to-Q amino acid change within the catalytic domain are set forth inFIG. 17. Similarly, the amino acid sequence of an aggrecanase-2 moleculewith an E-to-Q change at position 411 within the catalytic domain is setforth in FIG. 21 (SEQ ID NO: 30).

VII. Disease Treatment and Diagnosis

Inhibitors of aggrecanase activity may be used in the treatment ofdiseases described below. Inhibitors can also be used in the assays andmethods of detection described. Various diseases that are contemplatedas being treatable by using inhibitors of aggrecanases of the inventioninclude, but are not limited to, osteoarthritis, cancer, inflammatoryjoint disease, rheumatoid arthritis, septic arthritis, periodontaldiseases, corneal ulceration, proteinuria, coronary thrombosis fromatherosclerotic plaque rupture, aneurysmal aortic disease, inflammatorybowel disease, Crohn's disease, emphysema, acute respiratory distresssyndrome, asthma, chronic obstructive pulmonary disease, Alzheimer'sdisease, brain and hematopoietic malignancies, osteoporesis, Parkinson'sdisease, migraine, depression, peripheral neuropathy, Huntington'sdisease, multiple sclerosis, ocular angiogenesis, macular degeneration,aortic aneurysm, myocardial infarction, autoimmune disorders,degenerative cartilage loss following traumatic joint injury, headtrauma, dystrophobic epidermolysis bullosa, spinal cord injury, acuteand chronic neurodegenerative diseases, osteopenias, tempero mandibularjoint disease, demyelating diseases of the nervous system, organtransplant toxicity and rejection, cachexia, allergy, tissueulcerations, restenosis, and other diseases characterized by alteredaggrecanase activity or altered aggrecanase level.

Inhibitors and antibodies of the invention that inhibit activity ofaggrecanases and/or compounds that lower expression of aggrecanases maybe used in the treatment of any disease in a mammal that involvesdegradation of the extracellular matrix. An effective amount of at leastone of aggrecanase antibodies or inhibitors, in a pharmaceuticallyacceptable vehicle, can be used for treatment of diseases, such asosteoarthritis, or other diseases disclosed which are characterized bydegradation of matrix proteins, such as aggrecan, by aggrecanases andaggrecanase-related proteins.

VIII. Administration

Another aspect of the invention provides pharmaceutical compositionscontaining a therapeutically effective amount of an aggrecanaseinhibitor or antibody. Therefore, these compositions of the inventionmay be used in the treatment of diseases characterized by thedegradation of aggrecan by an aggrecanase enzyme or a protein withaggrecanase-like activity.

The invention includes methods for treating patients suffering fromconditions characterized by a degradation of aggrecan. These methods,according to the invention, entail administering to a patient needingsuch treatment an effective amount of a composition comprising anaggrecanase inhibitor which inhibits the proteolytic activity. It iscontemplated that inhibitors of the invention may function either byinhibiting aggrecanase activity or simply by regulating levels ofaggrecanases in a disease state.

Inhibitors of the present invention are useful to diagnose or treatvarious medical disorders in humans or animals. In one embodiment, theantibodies of the invention can be used to inhibit or reduce at leastone activity associated with an aggrecanase protein, relative to anaggrecanase protein not bound by the same antibody. In one embodiment,inhibitors of the invention can inhibit or reduce at least one of theactivities of an aggrecanase molecule relative to the aggrecanase thatis not bound by an antibody. In certain embodiments, an activity of anaggrecanase, when bound by at least one of the presently disclosedantibodies, is inhibited at least 50%, may be inhibited at least 60, 62,64, 66, 68, 70, 72, 72, 76, 78, 80, 82, 84, 86, or 88%, may be inhibitedat least 90, 91, 92, 93, or 94%, or may be inhibited at least 95% to100% relative to the aggrecanase protein that is not bound by at leastone of the presently disclosed antibodies.

Generally, compositions of the present are administered to a patient sothat antibodies or their binding fragments are administered at a doseranging from about 1 μg/kg to about 20 mg/kg, about 1 μg/kg to about 10mg/kg, about 1 μg/kg to about 1 mg/kg, about 10 μg/kg to about 1 mg/kg,about 10 μg/kg to about 100 μg/kg, about 100 μg to about 1 mg/kg, orabout 500 μg/kg to about 1 mg/kg. Antibodies are administered as a bolusdose, to maximize the interval of time that the antibodies can circulatein the patient's body following their administration to the patient.Continuous infusion may also be used after an initial bolus dose

In another embodiment, the invention is directed to administration ofinhibitors of aggrecanases, such as proteins, peptides, antibodies, andchemical compounds. The effective amount of an inhibitor is a dosagewhich is useful for reducing activity of aggrecanases to achieve adesired biological outcome. Generally, appropriate therapeutic dosagesfor administering an inhibitor may range, for example, from about 5 mgto about 100 mg, from about 15 mg to about 85 mg, from about 30 mg toabout 70 mg, or from about 40 mg to about 60 mg. Inhibitors can beadministered in one dose, or at intervals such as once daily, onceweekly, or once monthly. Dosage schedules for administration of anaggrecanase inhibitor can be adjusted based on, for example, theaffinity of the inhibitor for its aggrecanase target, the half-life ofthe inhibitor, and the severity of the patient's condition. Generally,inhibitors are administered as a bolus dose, to maximize theircirculating levels. Continuous infusions may also be used after thebolus dose.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell culture or experimentalanimal models, e.g., for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Inhibitors, which exhibit large therapeutic indices, are generallypreferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosages for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatexhibit an ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any inhibitor used according to the presentinvention, a therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose may be formulated in animal models toachieve a circulating plasma concentration range that exhibits an IC₅₀(i.e., the concentration of the test antibody which achieves ahalf-maximal inhibition of symptoms) as determined by cell cultureassays. Levels in plasma may be measured, for example, by highperformance liquid chromatography. The effects of any particular dosagecan be monitored by suitable bioassays. Examples of suitable bioassaysinclude DNA replication assays, transcription-based assays, GDFprotein/receptor binding assays, creatine kinase assays, assays based onthe differentiation of pre-adipocytes, assays based on glucose uptake inadipocytes, and immunological assays.

Therapeutic methods of the invention include administering anaggrecanase inhibitor composition topically, systemically, or locally asan implant or a device. The dosage regimen for the administration ofcomposition will be determined by the attending physician based onvarious factors which modify the action of the aggrecanase protein, thesite of pathology, the severity of disease, the patient's age, sex, anddiet, the severity of any inflammation, time of administration and otherclinical factors. Generally, systemic or injectable administration willbe initiated at a dose which is minimally effective, and the dose willbe increased over a preselected time course until a positive effect isobserved. Subsequently, incremental increases in dosage will be madelimiting to levels that produce a corresponding increase in effect,while taking into account any adverse affects that may appear. Theaddition of other known factors to a final composition may also affectthe dosage.

Progress can be monitored by periodic assessment of disease progression.The progress can be monitored, for example, by X-rays, MRI or otherimaging modalities, synovial fluid analysis, and/or clinicalexamination.

IX. Assays and Methods of Detection

The inhibitors and antibodies of the invention can be used in assays andmethods of detection to determine the presence or absence of, orquantify aggrecanase in a sample. The inhibitors and antibodies of thepresent invention may be used to detect aggrecanase proteins, in vivo orin vitro. By correlating the presence or level of these proteins with adisease, one of skill in the art can diagnose the associated disease ordetermine its severity. Diseases that may be diagnosed by the presentlydisclosed inhibitors and antibodies are set forth above.

Detection methods for use with antibodies are well known in the art andinclude ELISA, radioimmunoassay, immunoblot, western blot,immunofluorescence, immuno-precipitation, and other comparabletechniques. The antibodies may further be provided in a diagnostic kitthat incorporates at least one of these techniques to detect a protein(e.g., an aggrecanase protein). Such a kit may contain other components,packaging, instructions, or other material to aid the detection of anaggrecanase protein, and instructions regarding use of the kit. Whenprotein inhibitors, for example, peptide inhibitors, are used in suchdiagnostic assays, protein-protein interaction assays can be employed.

Where inhibitors are intended for diagnostic purposes, it may bedesirable to modify them, for example, with a ligand group (such asbiotin) or a detectable marker group (such as a fluorescent group, aradioisotope or an enzyme). If desired, the antibodies (whetherpolyclonal or monoclonal) may be labeled using conventional techniques.Suitable labels include fluorophores, chromophores, radioactive atoms,electron-dense reagents, enzymes, and ligands having specific bindingpartners. Enzymes are typically detected by their activity. For example,horseradish peroxidase can be detected by its ability to converttetramethylbenzidine (TMB) to a blue pigment, quantifiable with aspectrophotometer. Other suitable binding partners include biotin andavidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art.

The following examples illustrate practice of the present invention inisolating and characterizing human aggrecanase and otheraggrecanase-related proteins, obtaining the human proteins andexpressing the proteins via recombinant techniques.

EXAMPLES Example 1 Cloning of ADAMTS-5 (Aggrecanase-2)

PCR primers were designed to the published sequence for human ADAMTS-5(GenBank Accession #AF142099). The full-length coding sequence forADAMTS-5 was amplified from a human uterus cDNA library (GeneticsInstitute/Wyeth Research) using the Advantage-GC PCR kit (Clontech).

ADAMTS-5 was isolated using PCR. Tissue expression pattern ofaggrecanase-5 was determined by PCR amplification of 7 different oligodT-primed human cDNA libraries including placenta, brain, muscle, lung,heart, uterus, and spinal cord. PCR primers that amplified 5′ and 3′regions of the ADAMTS-5 cDNA were used. Primer sequences for amplifyingthe 5′ portion of ADAMTS-5 were as follows: 5′ primer:5′GACTGACTGAATTCATACCCATAAAGTCCCAGTCGCGCA (SEQ ID NO: 16), whichincorporated an 8 bp tail (GACTGACT), and an EcoR1 site (GAATTC)upstream of the start codon (ATG) of the ADAMTS-5 sequence and 3′primer: 5′ CAGGGCTTAGATGCATCAATGCTGG (SEQ ID NO: 17). Primer sequencesfor amplifying 3′ portion of ADAMTS-5 were as follows: 5′ primer: 5′TACCAGCATTGATGCATCTAAGCCCT (SEQ ID NO: 18) and 3′ primer 5′AAATGGGCGCGGCCGCTGCATCGGTGCTGAATCCTCCAGTTATCT (SEQ ID NO: 19), whichincorporated an 8 bp tail (AAATGGGC) and a Not1 site (GCGGCCGC)downstream of the stop codon (TAG) for ADAMTS-5. PCR products of theappropriate size, 5′ amplification product of 1404 bp and 3′amplification product of 1519 bp were found using a uterus cDNA libraryas substrate. The Advantage-GC PCR Kit from Clontech was used for thePCR reactions. Reaction conditions were those recommended by themanufacturer, with the following exceptions: the amount of GC Melt usedwas 10 μl per 50 μl reaction; the amount of Not1 linearized library usedwas 0.2 ng/μl reaction; and the amount of each oligo used was 2 pmol/ulreaction. Cycling conditions were as follows: 95° C. for 1 min, onecycle; followed by 30 cycles consisting of 95° C. for 15 sec/68° C. for2 min. The 2 overlapping PCR products resulting from the amplificationswere digested with EcoR1 and Nsi1 (5′ product) or Nsi1 and Not1 (3′product) and ligated into the CHO expression vector pHTop_new, digestedwith EcoR1 and Not1 using standard ligation enzyme, buffers andconditions. Ligated products were used to transform ElectroMAX DH10Bcells from Life Technologies (Carlsbad, Calif.). Cloned PCR fragments ofADAMTS-5 were sequenced to verify sequence.

Nucleotide sequence for full-length ADAMTS-5 protein was the consensussequence derived from the PCR products. Two silent changes werereflected in this sequence as compared to the published sequence forADAMTS-5. These changes were a G to an A at nucleotide #711 and an A toa G at nucleotide #2046 (numbering starts at 1 for the A of the ATGstart codon for ADAMTS-5). The full-length cDNA, including an openreading frame (ORF) for ADAMTS-5 ORF and 5′ and 3′ untranslated regions(UTRs), was subcloned into the mammalian expression vector pED6-dpc2.

A cDNA expressing the full-length human aggrecanase-2 (hAgg-2)/ADAMTS-5protein was cloned into the expression plasmid pHTop. This plasmid wasderived from pED (Kaufman et al., Nucleic Acids Res. 19:4485–4490 (1991)by removing the majority of the adenomajor late promoter and insertingsix repeats of the tet operator (Gossen and Bujard, Proc. Natl. Acad.Sci. U.S.A. 89:5547–5551 (1992)). A CHO cell line stably expressinghAgg-2 was obtained by transfecting pHTop/hAgg-2 into CHO/A2 cells andselecting clones in 0.05 μM methotrexate. Clones were screened forAggrecanase-2 expression by western analysis of conditioned media usinga polyclonal antibody specific for Aggrecanase-2. The CHO/A2 cell linewas derived from CHO DUKX B11 cells (Urlaub and Chasin, Proc. Natl.Acad. Sci. U.S.A. 77:4216–4220 (1980)) by stably integrating atranscriptional activator, a fusion between the tet repressor and theherpes virus VP16 transcription activation domain (Gossen and Bujard,Proc. Natl. Acad. Sci. U.S.A. 89:5547–5551 (1992)).

A truncated ADAMTS-5 protein including a C-terminal protein purificationtag was constructed using 3 DNA fragments from the full-length ADAMTS-5construct described above and a synthetic DNA duplex. ADAMTS-5 wastruncated at amino acid residue 752 (Pro) of the full-length aggrecanaseprotein. Proline at 752 in the full-length protein is N-terminal of acleavage site in the full-length protein. The 3 DNA fragments consistedof a 5777 bp SgrA1/Not1 fragment containing the pHTop_new vectorbackbone and a 5′ portion of ADAMTS-5, a 1756 bp SgrA1/BspH1 fragmentcontaining ADAMTS-5, and a 304 bp BspH1/BsrG1 fragment containingADAMTS-5. The 4 oligonucleotide fragments used to build the 110 bpsynthetic duplex were as follows: oligonucleotide fragment 1:5′GTACAAAGATTGTTGGAACCTTTAATAAGAAAA GTAAGGGTTACACTGACGTGGTGAGGATTC (SEQID NO: 20); oligonucleotide fragment 2: 5′CTGGATCCGGATCTGCTTGGAGCCACCCGCAGTTCGAAA AATAAGGC (SEQ ID NO: 21) whichencodes a GSGSA (SEQ ID NO: 22) peptide linker followed by a WSHPQFEK(SEQ ID NO: 23) Strep-tag II protein purification tag from IBA fusedC-terminus to amino acid 752 of the truncated ADAMTS-5 protein,oligonucleotide fragment 3: 5′ GGCCGCCTTATTTTTCGAACTGCGGGTGGCTCCAAGCAGATCCGGATCCAGGAATCCTCAC (SEQ ID NO: 24) and oligonucleotidefragment 4: 5′ CACGTCAGTGTAACCCTTACTTTTCTTATTAAAGGTTCCAACA ATCTTT (SEQID NO: 25). Each oligonucleotide fragment was diluted to a finalconcentration of 10 pmol/μl with sterile water. Equal volumes (10 μleach) of oligonucleotide fragments 1 and 4 were mixed. Equal volumes (10μl each) of oligonucleotide fragments 2 and 3 were mixed. Mixtures wereheated to 95° C. for 5 minutes and then allowed to cool to roomtemperature. 1 μl of each mixture was used in the final ligation withthe 3 ADAMTS-5 DNA fragments described above. Standard ligation enzyme,buffers, and conditions were used. Ligated products were used totransform ElectroMAX DH10B cells from Life Technologies (Carlsbad,Calif.). The cloned synthetic oligonucleotides were sequenced todetermine fidelity and verify sequence. FIG. 18 is a schematicrepresentation of Streptavidin-tagged aggrecanase-2 proteins of theinvention.

Example 2 Expression of ADAMTS-5-wild type (WT) and Active Site Mutant(ASM)

CHO/A2 cells were transfected with ADAMTS-5-WT or ASM expression vectorsusing Lipofectin (Life Technologies, Inc.). The cells were plated in thepresence of 0.02, 0.05, and 0.1 mM Methotrexate (MTX) selection andincubated at 37° C., 5% CO₂ for 2 weeks. Colonies were picked andexpanded into cell lines while cultured in selection medium.

In another experiment, CHO cells were transfected with active sitemutants for aggrecanase-1, as depicted in FIG. 17.

Example 3 Subcloning of ADAMTS-5 Active Site Mutant

The E411-Q411 point mutation in ADAMTS-5 was generated using theQuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla,Calif.). Mutagenesis was performed on pHTop/Agg-2 using the QuickChangeSite Directed Mutation Kit (Stratagene, catalog #200518). The singlebase pair mutation, 2597-G to 2597-C, resulted in a single amino acidchange at position 411 (E-to-Q) in the catalytic domain of aggrecanase-2protein (SEQ ID NO: 30). This mutation was shown to inactivate thecatalytic activity of mini-stromelysin-1 (Steele et al., ProteinEngineering 13:397–405(2000)). A CHO cell line stably expressinghAgg-2(E411Q) active site mutant was obtained by transfectingpHTop/hAgg-2(E411Q) into CHO/A2 cells and selecting clones in 0.05 μMmethotrexate. Clones were screened for Aggrecanase-2 expression bywestern analysis of conditioned media using a polyclonal antibodyspecific for Aggrecanase-2.

Additionally, E-to-Q mutation in the active site of aggrecanase-1molecules of the invention is generated. FIG. 17 is a schematicrepresentation of recombinant truncated aggrecanase-1 molecules of theinvention that include an E-to-Q mutation in the catalytic domain.Nucleic acid sequences encoding these aggrecanase-1 molecules are clonedinto an appropriate vector, for example, pHTop disclosed, andsubsequently transfected into an appropriate cell line, for example, CHOcells. Stable transfectants for aggrecanases of the invention can beselected as described above. Expression levels of active siteaggrecanase mutants can be detected using an antibody specific for theaggrecanase being expressed.

Example 4 Western Blotting

Conditioned medium from CHO cells expressing ADAMTS-5 WT or ASM wereloaded on a 12% SDS-PAGE gel under reducing conditions. The samples werethen transferred to a nitrocellulose membrane. ADAMTS-5 protein wasdetected by a polyclonal antibody against ADAMTS-5, followed bygoat-anti-rabbit IgG-HRP and a chemiluminescent substrate (Pierce,Milwaukee, Wis.).

In another experiment, conditioned medium from CHO cells expressingtruncated aggrecanase-2 proteins was loaded onto a 12% SDS-PAGE. Theresults of such an experiment are shown in FIG. 19. Briefly, CHO cellswere transfected with a nucleic acid encoding either truncatedaggrecanase-2 from amino acid #1 through amino acid #567, or truncatedaggrecanase-2 from amino acid #1 through amino acid #628, or a nucleicacid expressing a full-length aggrecanase-2 molecule. Stable CHO celllines were developed expressing either aggrecanase-2 from amino acid #1through #567, or from amino acid #1 through #628, or the full-lengthaggrecanase-2 molecule. Cells were subsequently harvested for proteinsand expression levels for the various aggrecanase-2 molecules wasdetermined by Western blot analysis using an antibody specific toaggrecanase-2. As demonstrated by the experiment set forth in FIG. 19,truncated aggrecanase proteins are expressed at higher levels, due tohigher stability, as compared with the full-length aggrecanase-2protein.

Example 5 Microcapillary HPLC-Mass Spectrometry

Recombinant ADAMTS-5-WT or ASM proteins were purified by HP-HPLC andfurther analyzed by 1D-SDS-polyacrylamide gel electrophoresis. Proteinswere visualized by Coommassie blue staining, and protein bands ofinterest were excised manually, then reduced, alkylated and digestedwith trypsin or endopeptidase Lys-C (Promega, Madison, Wis.) in situusing an automated in-gel digestion robot. After digestion, the peptideextracts were concentrated and separated by microelectrosprayreverse-phase HPLC. Peptide analyses were done on a Finnigan LCQ iontrap mass spectrometer (ThermoQuest, San Jose, Calif.). Automatedanalysis of MS/MS data was performed using the SEQUEST computeralgorithm incorporated into the Finnigan Bioworks data analysis package(ThermoQuest, San Jose, Calif.) using the database of proteins derivedfrom the complete genome.

Example 6 Biological Activity of Expressed Aggrecanase

To measure the biological activity of the expressed aggrecanaseproteins; for example, truncated aggrecanases of the inventiondisclosed, the proteins are recovered from the cell culture and purifiedby isolating the aggrecanase-related proteins from other proteinaceousmaterials with which they are co-produced as well as from othercontaminants. Purification is carried out using standard techniquesknown to those skilled in the art. The isolated protein may be assayedin accordance with the following assays:

Assays specifically to determine if the protein is an enzyme capable ofcleaving aggrecan at the aggrecanase cleavage site:

Fluorescent peptide assay: Expressed protein is incubated with asynthetic peptide which encompasses amino acids at the aggrecanasecleavage site of aggrecan. Either the N-terminus or the C-terminus ofthe synthetic peptide is labeled with a fluorophore and the otherterminus includes a quencher. Cleavage of the peptide separates thefluorophore and quencher and elicits fluorescence. From this assay it isdetermined that the expressed aggrecanase protein can cleave aggrecan atthe aggrecanase site and that relative fluorescence is a determinationof the relative activity of the expressed protein.

Neoepitope western: Expressed aggrecanase protein is incubated withintact aggrecan. After several biochemical manipulations of theresulting sample (dialysis, chondroitinase treatment, lyophilization,and reconstitution) the sample is run on an SDS PAGE gel. The gel isincubated with an antibody that is specific to a site on aggrecan whichis only exposed after aggrecanase cleavage. The gel is transferred ontonitrocellulose paper and developed using a secondary antibody (called awestern assay) which subsequently results in a banding patternindicative of products with a molecular weight consistent withaggrecanase generated cleavage products of aggrecan. This assay resultsin the finding that the expressed aggrecanase protein cleaved nativeaggrecan at the aggrecanase cleavage site, and also gives the molecularweight of the cleavage products. Relative density of the bands can givean indication of relative aggrecanase activity.

Assay to determine if an expressed protein can cleave aggrecan anywherein the protein (not specific to the aggrecanase site).

Aggrecan ELISA: Expressed protein is incubated with intact aggrecanwhich had been previously adhered to plastic wells. The wells are washedand then incubated with an antibody that detects aggrecan. The wells aredeveloped with a secondary antibody. If the original amount of aggrecanremains in the wells, the antibody staining is dense. Whereas, ifaggrecan was digested by aggrecanase activity of the expressedaggrecanase protein, the aggrecan comes off the plate and the subsequentstaining of the aggrecan-coated wells by the antibody is reduced. Thisassay tells whether an expressed protein is capable of cleaving aggrecan(anywhere in the protein, not only at the aggrecanase site) and canfurther determine relative aggrecan cleavage.

The results of one such experiment are shown in FIG. 20, where aggrecancleavage was detected after incubation of aggrecan with truncatedaggrecanase-2 molecules using a 3B3 ELISA assay. The results of such anexperiment, shown in FIG. 20 include % aggrecan cleavage subsequent toincubation with truncated aggrecanases, for example, aggrecanase-2 fromamino acid #1 through #567 and aggrecanase-2 from amino acid #1 through#628. As shown in FIG. 20, truncated aggrecanases of the inventionhaving one or both TSP domains deleted are biologically active.

Protein analysis of isolated proteins is conducted using standardtechniques such as SDS-PAGE acrylamide (Laemmli, Nature 227:680–685(1970)) stained with silver (Oakley, et al., Anal Biochem. 105:361–363(1980)) and by immunoblot (Towbin, et al., Proc. Natl. Acad. Sci. USA76:4350–4354 (1979)). Using the above described assays, expressedaggrecanase-related proteins are evaluated for their activity and usefulaggrecanase-related molecules are identified.

Activity Assay: Mirotiter plates (Costar) were coated with hyaluronicacid (ICN), followed by chondroitinase (Seikagaku Chemicals)-treatedbovine aggrecan. Conditioned medium from CHO cells expressing WTADAMTS-5 or ADAMTS-5 ASM was added to the aggrecan-coated plates.Aggrecan cleaved at the E³⁷³-A³⁷⁴ within the interglobular domain waswashed away. The remaining uncleaved aggrecan was detected with the 3B3antibody (ICN), followed by anti-IgM-HRP secondary antibody (SouthernBiotechnology). Final color development was With3,3″,5,5″tetramethylbenzidine (TMB, BioFx Laboratories).

ADAMTS-5 is synthesized in the inactive pro-form (˜90 kDa) and can beprocessed by furin to yield the mature species of ˜70 kDa. Conditionedmedium from CHO lines transfected with ADAMTS-5 expressed a small amountof active, mature protein. The predominant species expressed was aprotein of ˜55 kDa, representing a cleavage product of the matureprotein.

Conditioned medium from the CHO stable lines transfected with ADAMTS-5showed that the enzyme was cleaved to yield a species of ˜55 kDa. Massspectrometry and N-terminal sequencing of the clipped form revealed thatthe cleavage occurred between E⁷⁵³-G⁷⁵⁴ residues in the spacer domain.In the presence of EDTA or a non-specific hydroxamate metalloproteaseinhibitor, the full-length mature protein, ˜70 kDa, was preserved. Thiscleavage is autocatalytic and not due to an enzyme present in the CHOconditioned medium. Site-specific mutagenesis was performed to mutatethe E⁴¹¹-Q⁴¹¹ within the catalytic HELGH motif (SEQ ID NO: 26) ofADAMTS-5. Stable CHO lines were made with this ADAMTS-5 activesite-mutant (ADAMTS-5 ASM). Conditioned medium from CHO stable linestransfected with ADAMTS-5 ASM lacked aggrecanase activity as shown byELISA (FIG. 16). The full-length ˜70 kDa protein rather than the clipped55 kDa form, was predominant in a western blot of conditioned media fromCHO cells expressing ADAMTS-5 ASM.

The above described examples demonstrate that recombinant ADAMTS-5 wassusceptible to proteolytic cleavage at residue E⁷⁵³-G⁷⁵⁴ in the spacerdomain. This cleavage reduced the size of the mature protein to ˜55 kDaand was inhibited by EDTA and a non-specific hydroxamate metalloproteaseinhibitor. A point mutation in the catalytic domain of ADAMTS-5inactivated the enzymatic activity of the protein and protected thefull-length protein from cleavage. It is therefore contemplated that theproteolytic processing was autocatalytic and not due to a proteasepresent in the conditioned medium of the CHO cells.

Example 7 Expression of Aggrecanase

In order to produce murine, human, or other mammalianaggrecanase-related proteins, the DNA encoding it is transferred into anappropriate expression vector and introduced into mammalian cells orother preferred eukaryotic or prokaryotic hosts including insect hostcell culture systems by conventional genetic engineering techniques.Expression system for biologically active recombinant human aggrecanaseis contemplated to be stably transformed mammalian cells, insect, yeast,or bacterial cells.

One skilled in the art can construct mammalian expression vectors byemploying the nucleotide sequence of FIG. 3 from nucleotide #1 through#2259; nucleotide sequence of FIG. 5 from nucleotide #1 throughnucleotide #2256; nucleic acid sequence of FIG. 7 from nucleotide #1through nucleotide #1884; or nucleic acid sequence of FIG. 9 fromnucleotide #1 through nucleotide #1701 or other DNA sequences encodingaggrecanases or aggrecanase-like proteins or other modified sequencesand known vectors, such as pCD (Okayama and Berg, Mol. Cell Biol.,2:161–170 (1982)), pJL3, pJL4 (Gough et al., EMBO J., 4–645–653 (1985))and pMT2 CXM.

The mammalian expression vector pMT2 CXM is a derivative of p91023(b)(Wong et al., Science 228:810–815, 1985) differing from the latter inthat it contains the ampicillin resistance gene in place of thetetracycline resistance gene and further contains a XhoI site forinsertion of cDNA clones. The functional elements of pMT2 CXM have beendescribed (Kaufman, R. J., Proc. Natl. Acad. Sci. USA 82:689–693 (1985))and include the adenovirus VA genes, the SV40 origin of replicationincluding the 72 bp enhancer, the adenovirus major late promoterincluding a 5′ splice site and the majority of the adenovirus tripartiteleader sequence present on adenovirus late mRNAs, a 3′ splice acceptorsite, a DHFR insert, the SV40 early polyadenylation site (SV40), andpBR322 sequences needed for propagation in E. coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which hasbeen deposited with the American Type Culture Collection (ATCC),Rockville, Md. (USA) under accession number ATCC 67122. EcoRI digestionexcises the cDNA insert present in pMT2-VWF, yielding pMT2 in linearform which can be ligated and used to transform E. coli HB 101 or DH-5to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis (Morinaga et al., Biotechnology 84: 636–639 (1984)). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts the following sequence:

-   -   5′ PO-CATGGGCAGCTCGAG 3′ (SEQ ID NO:27) at nucleotide #1145.        This sequence contains the recognition site for the restriction        endonuclease Xho I. A derivative of pMT2CXM, termed pMT23,        contains recognition sites for the restriction endonucleases        PstI, EcoRI, SalI, and XhoI. Plasmid pMT2 CXM and pMT23 DNA may        be prepared by conventional methods.

pEMC2β1 derived from pMT21 may also be suitable in practice of theinvention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. Asdescribed above EcoRI digestion excises the cDNA insert present inpMT-VWF, yielding pMT2 in linear form which can be ligated and used totransform E. Coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2DNA can be prepared by conventional methods.

pMT21 is derived from pMT2 through the following two modifications.First, 76 bp of the 5′ untranslated region of the DHFR cDNA including astretch of 19 G residues from G/C tailing for cDNA cloning is deleted.In this process, a XhoI site is inserted to obtain the followingsequence immediately upstream from DHFR:

(SEQ ID NO:28) 5′-CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG                        PstI      EcoRI XhoI

Second, a unique ClaI site is introduced by digestion with EcoRV andXbaI, treatment with Klenow fragment of DNA polymerase I, and ligationto a ClaI linker (CATCGATG). This deletes a 250 bp segment from theadenovirus associated RNA (VAI) region but does not interfere with VAIRNA gene expression or function. pMT21 is digested with EcoRI and XhoI,and used to derive the vector pEMC2B1.

A portion of the EMCV leader is obtained from pMT2-ECAT1 (Jang et al.,J. Virol 63:1651–1660 (1989)) by digestion with EcoRI and PstI,resulting in a 2752 bp fragment. This fragment is digested with TaqIyielding an EcoRI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5′ TaqI protruding end and a3′ XhoI protruding end which has the following sequence:

(SEQ ID NO:29) 5′CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTT   TaqI CCTTTGAAAAACACGATTGC 3′         XhoI

This sequence matches the EMC virus leader sequence from nucleotide #763to #827. It also changes the ATG at position 10 within the EMC virusleader to an ATT and is followed by a XhoI site. A three way ligation ofthe pMT21 EcoRI-16hoI fragment, the EMC virus EcoRI-TaqI fragment, andthe 68 bp oligonucleotide adapter TaqI-16hoI adapter resulting in thevector pEMC2β1.

This vector contains the SV40 origin of replication and enhancer, theadenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

The construction of vectors may involve modification of theaggrecanase-related DNA sequences. For instance, a cDNA encoding anaggrecanase can be modified by removing the non-coding nucleotides onthe 5′ and 3′ ends of the coding region. The deleted non-codingnucleotides may or may not be replaced by other sequences known to bebeneficial for expression. These vectors are transformed intoappropriate host cells for expression of aggrecanase or aggrecanase-likeproteins. Additionally, the sequence of FIG. 3 from nucleotide #1through nucleotide #2259, or FIG. 5 from nucleotide #1 throughnucleotide #2256, or FIG. 7 from nucleotide #1 through nucleotide #1884,or FIG. 9 from nucleotide #1 through nucleotide #1701, or fragments ofvariants thereof or other sequences encoding aggrecanases of theinvention or proteins that have aggrecanase activity can be manipulatedto express an aggrecanase or aggrecanase-like protein by deletingaggrecanase encoding propeptide sequences and replacing them withsequences encoding the complete propeptides of other aggrecanaseproteins.

One skilled in the art can manipulate the sequences of FIG. 3 fromnucleotide #1 through nucleotide #2259; or FIG. 5 from nucleotide #1through nucleotide #2256; or FIG. 7 from nucleotide #1 throughnucleotide #1884; or FIG. 9 from nucleotide #1 through nucleotide #1701;or nucleotide sequences encoding aggrecanases molecules of FIGS. 12 and13 set forth in FIGS. 23 and 24, or fragments and variants thereof, byeliminating or replacing the mammalian regulatory sequences flanking thecoding sequence with bacterial sequences to create bacterial vectors forintracellular or extracellular expression of truncated aggrecanasemolecules. For example, the coding sequences could be furthermanipulated (e.g. ligated to other known linkers or modified by deletingnon-coding sequences therefrom or altering nucleotides therein by otherknown techniques). A modified aggrecanase encoding sequence could thenbe inserted into a known bacterial vector using procedures such asdescribed in T. Taniguchi et al., Proc. Natl Acad. Sci. USA,77:5230–5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells to express an aggrecanase proteinof the invention. For a strategy for producing extracellular expressionof aggrecanase-related proteins in bacterial cells, see, e.g. Europeanpatent application-EP 177,343.

Similar manipulations can be performed for construction of an insectvector (see, e.g., procedures described in published European patentapplication EP 155,476) for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. (See, e.g., procedures described in publishedPCT application WO 86/00639 and European patent application EP 123,289).

A method for producing high levels of an aggrecanase-related protein ofthe invention in mammalian, bacterial, yeast, or insect host cellsystems may involve the construction of cells containing multiple copiesof the heterologous aggrecanase-related gene. The heterologous gene islinked to an amplifiable marker, e.g., the dihydrofolate reductase(DHFR) gene for which cells containing increased gene copies can beselected for propagation in increasing concentrations of methotrexate(MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol.,159:601–629 (1982). This approach can be employed with a number ofdifferent cell types.

For example, a plasmid containing a DNA sequence for an aggrecanase oraggrecanase-like protein of the invention in operative association withother plasmid sequences enabling expression thereof and the DHFRexpression plasmid pAdA26SV(A)3 (Kaufman and Sharp, Mol. Cell. Biol.,2:1304–1319 (1982)) can be co-introduced into DHFR-deficient CHO cells,DUKX-BII, by various methods including calcium phosphate coprecipitationand transfection, electroporation or protoplast fusion. DHFR expressingtransformants are selected for growth in alpha media with dialyzed fetalcalf serum, and subsequently selected for amplification by growth inincreasing concentrations of MTX (e.g. sequential steps in 0.02, 0.2,1.0 and 5 μM MTX) as described in Kaufman et al., Mol Cell Biol.,5:1750–1759 (1985). Transformants are cloned, and biologically activeaggrecanase expression is monitored by at least one of the assaysdescribed above. Aggrecanase protein expression should increase withincreasing levels of MTX resistance. Aggrecanase polypeptides arecharacterized using standard techniques known in the art such as pulselabeling with ³⁵S methionine or cysteine and polyacrylamide gelelectrophoresis. Similar procedures can be followed to produce otheraggrecanases or aggrecanase-like proteins.

In one example, an aggrecanase nucleotide sequence of the presentinvention is cloned into the expression vector pED6 (Kaufman et al.,Nucleic Acid Res. 19:4485–4490 (1991)). COS and CHO DUKX B11 cells aretransiently transfected with an aggrecanase sequence of the invention(+/−co-transfection of PACE on a separate pED6 plasmid) by lipofection(LF2000, Invitrogen). Duplicate transfections are performed for eachmolecule of interest: (a) one transfection set for harvestingconditioned media for activity assay and (b) one transfection set for35-S-methionine/cysteine metabolic labeling.

On day one media is changed to DME(COS) or alpha (CHO) media+1%heat-inactivated fetal calf serum+/−100 μg/ml heparin on wells of set(a) to be harvested for activity assay. After 48 h (day 4), conditionedmedia is harvested for activity assay.

On day 3, the duplicate wells of set (b) were changed to MEM(methionine-free/cysteine free) media+1% heat-inactivated fetal calfserum+100 μg/ml heparin+100 μCi/ml 35S-methionine/cysteine (Redivue Promix, Amersham). Following 6 h incubation at 37° C., conditioned media isharvested and run on SDS-PAGE gels under reducing conditions. Proteinsare visualized by autoradiography.

Example 8 Biological Activity of Expressed Aggrecanase

To measure the biological activity of the expressed aggrecanase-relatedproteins obtained in the examples above, the proteins are recovered fromthe cell culture and purified by isolating the aggrecanase-relatedproteins from other proteinaceous materials with which they areco-produced as well as from other contaminants. The purified protein maybe assayed in accordance with assays described above. Purification iscarried out using standard techniques known to those skilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGEacrylamide (Laemmli, Nature 227:680–685 (1970)) stained with silver(Oakley et al., Anal. Biochem. 105:361–363 (1980)) and by immunoblot(Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350–4354 (1979)).

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

1. A nucleic acid encoding a fusion protein, the nucleic acidcomprising: a nucleic acid encoding an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 4, 6, and 8; a nucleic acid encodingat least one peptide tag; and a nucleic acid encoding at least onepeptide linker connecting the nucleic acid encoding the amino acidsequence to the nucleic acid encoding the at least one peptide tag. 2.The nucleic acid of claim 1, wherein the amino acid sequence is SEQ IDNO:4.
 3. The nucleic acid of claim 1, wherein the amino acid sequence isSEQ ID NO:6.
 4. The nucleic acid of claim 1, wherein the amino acidsequence is SEQ ID NO:8.
 5. A truncated nucleic acid encoding anaggrecanase consisting of an amino acid sequence at least 95% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs: 4, 6, and 8, or fragments thereof, wherein said fragments retainaggrecanase activity.
 6. A nucleic acid sequence encoding a fusionprotein, the nucleic acid comprising: the truncated nucleic acid ofclaim 5, a nucleic acid encoding at least one peptide tag.
 7. Thenucleic acid of claim 5, wherein the aggrecanase consists of the aminoacid sequence of SEQ ID NO:4.
 8. The nucleic acid of claim 5, whereinthe aggrecanase consists of the amino acid sequence of SEQ ID NO:6. 9.The nucleic acid of claim 5, wherein the aggrecanase consists of theamino acid sequence of SEQ ID NO:8.
 10. The nucleic acid of claim 6,wherein the amino acid sequence is SEQ ID NO:4.
 11. The nucleic acid ofclaim 6, wherein the amino acid sequence is SEQ ID NO:6.
 12. The nucleicacid of claim 6, wherein the amino acid sequence is SEQ ID NO:8.